CN108503696B

CN108503696B - Zika virus subunit vaccine expressed by yeast cells

Info

Publication number: CN108503696B
Application number: CN201710108477.5A
Authority: CN
Inventors: 黄忠; 张伟; 屈攀科; 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: 2023-05-12
Anticipated expiration: 2037-02-27
Also published as: CN108503696A

Abstract

The invention provides a subunit vaccine of Zika virus expressed by yeast cells, in particular to a subunit Zika virus vaccine developed by utilizing the yeast cells, which has the advantages of high yield, high purity, good stability and easy purification, and meanwhile, as the subunit vaccine does not contain viral nucleic acid components, the subunit vaccine has no possibility of restoring mutation and is high in safety.

Description

Zika virus subunit vaccine expressed by yeast cells

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a Zika virus subunit vaccine expressed by yeast cells.

Background

Zika virus belongs to the Flaviviridae (Flaviviridae) genus of Flaviviridae (Flaviviruses) in the biological classification, which is a single positive strand RNA virus. Zika virus was first isolated from Uganda Zhai Carsen macaque since 1947 and was not noticed until 2013, and it was found that outbreaks of Guillain Bay syndrome were associated with the prevalence of French Bolitinia Zhai card virus. The Zika virus is mainly transmitted through the bite of aedes mosquitoes, and although most infections are asymptomatic, more and more small head disease cases are found to be related to the infection of the Zika virus during pregnancy of a mother, and the relationship between the two is further confirmed by the Zika virus separated from amniotic fluid and brain tissue of the small head disease fetus. Outbreaks of Zika virus pose a serious threat to public health worldwide.

Thus, there is a strong need in the art to develop a zika virus vaccine and a suitable method of preparing the same in order to effectively and purposefully 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 invention, there is provided an antigenic peptide derived from the envelope protein of the zika virus and selected from the group consisting of:

(1) An amino acid sequence shown in SEQ ID NO. 1;

(2) A derivative polypeptide formed by substitution, deletion or addition of one or more (less than or equal to 20, such as 2-10, preferably 2-5) amino acid residues to the amino acid sequence shown in SEQ ID NO.1, wherein the derivative polypeptide has the functions of inhibiting the cell infection of Zika virus and/or inducing an immune response against Zika virus.

In another preferred embodiment, the antigenic peptide is a recombinant protein expressed by a yeast cell.

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

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

(a) A polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

(b) A polynucleotide with a sequence shown as SEQ ID NO. 3;

(c) A polynucleotide having a nucleotide sequence having a homology of 95% (preferably 98%) or more with the sequence shown in SEQ ID NO. 3;

(d) A polynucleotide of 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added at the 5 'and/or 3' end of the polynucleotide shown in SEQ ID NO. 3;

(e) A polynucleotide complementary to the polynucleotide of any one 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 a prokaryotic cell and a eukaryotic cell.

In another preferred embodiment, the host cell comprises yeast, drosophila S2 cell, E.coli, CHO cell, DC cell, etc.

In a fifth aspect of the invention, there is provided a pharmaceutical 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, together with a pharmaceutically acceptable carrier and/or adjuvant.

In another preferred embodiment, the composition is a 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, together with 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, quick A, muramyl dipeptide, mineral or vegetable oil, vesicle-based adjuvants, nonionic block copolymers 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 as described in the first aspect of the invention, (a) for the preparation of an antibody against a zika virus; and/or (b) for preparing a medicament for treating and/or preventing diseases related to Zika virus.

In another preferred embodiment, the disease associated with Zika virus comprises: zika virus infection, guillain Barre syndrome, microcephaly, and the like.

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, in step (i) of the method, transformed yeast single colonies are inoculated into BMGY medium, the supernatant is removed by centrifugation after culturing, the cells are resuspended in BMMY (containing 1% methanol) medium, and the culture is induced for 24 to 72 hours (preferably 48 hours) at 25 to 35 ℃ (preferably 30 ℃).

In a ninth aspect of the invention there is provided a method of treatment by 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 understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows plasmid maps of pPink. Alpha. -HC-ZIKV-ED III and pPink. Alpha. -HC-ZIKV-E80.

FIG. 2 shows a small selection of Pichia pastoris high expression strains. (A) The culture medium parts of different clones are taken from 5ul samples of SDS-PAGE running gel, and the target protein is detected by Western-blot, and the size of the target protein (ZIKA-ED III and His-tag are fused) is 12.74kDa. (B) The culture medium parts of different clones are taken to obtain 10ul samples of SDS-PAGE running gel, and the target protein (ZIKA-E80 and His-tag fusion) is detected by Western-blot, and the size of the target protein (ZIKA-E80 and His-tag fusion) is 45.39kDa. Control (ctr) is a clone obtained from electrolinearized pPink. Alpha. -HC.

FIG. 3 shows the expression and purification of ZIKA-ED III in Pichia pastoris. (A) SDS-PAGE and Coomassie Brilliant blue staining analysis of purified ZIKA-ED III. (B) Western-blot detection was performed using antibodies against ZIKV-E80 (left) and His tag (right).

FIG. 4 shows that ZIKA-ED III inhibits infection of Vero cells by ZIKA virus. The purified ZIKA-ED III was diluted in series, mixed with 100PFU ZIKA virus, immediately added to the pre-spread Vero cells, cultured in a 37℃CO2 incubator for one hour, then the mixture of ZIKA-ED III and ZIKA virus was replaced with 2% DMEM medium containing 0.2% agarose, placed in a 37℃CO2 incubator for culturing, and after plaque formation, fixed with 4% paraformaldehyde and stained with crystal violet. BSA served as a negative control. (A) Reduction in the number of plaques in cells treated with ZIKA-ED iii compared to cells treated with BSA. (B) quantitative analysis of normalized plaque reduction. Mean ± standard error has been noted.

FIG. 5 shows the induction of neutralizing antibodies by ZIKA-ED III in balb/c mice. (A) ZIKA-ED III immunized balb/c mice. Mouse serum was collected two weeks after the second and two weeks after the third, respectively, and at a dilution of 1:10000, the antibody response specific for ZIKA-ED iii was detected by ELISA. (B) Plaque reduction neutralization experiments in serum two weeks after three days, 24-well plate crystal violet staining sections were shown. (C) data analysis of PRNT50 s. The geometric mean and p-values of PRNT50s are shown.

Figure 6 shows that EDIII antisera have in vivo protective effects against ZIKV infection. Two groups of five week old AG6 mice were set, five in each group. Antisera 50 ul/was mixed with an equal volume of 5PFU virus-containing dilution alone and incubated at 37℃for one hour. Serum virus mixtures were then intraperitoneally injected and weight changes and survival were recorded for two consecutive weeks. Mice lost 20% of their body weight over their original body weight, and were euthanized, defined as dead, and the body weight on the day was no longer recorded.

Detailed Description

Through extensive and intensive studies, the present inventors have unexpectedly found that subunit Zika virus vaccines developed using yeast cells have the advantages of high yield, high purity, good stability, and easy purification, and simultaneously, since they do not contain viral nucleic acid components, there is no possibility of restoring mutation, and safety is high. Furthermore, it was found experimentally that in combination with aluminium adjuvants, neutralizing antibodies induced with very low doses of immunogen (ZIKV EDIII) are sufficient to protect AG6 mice from lethal dose of zika virus. The in vivo and in vitro experimental results show that the ZiKV subunit vaccine ZIKV EDIII provided by the invention is a better vaccine for preventing Zika virus infection, and has a remarkably better protection effect.

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods 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, as 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, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (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 described herein.

Zika virus envelope proteins

The envelope protein (E protein) of the zika virus is the main target of neutralizing antibodies, whereas the E protein is divided into three regions, EDI, EDII, EDIII, most specific antibodies or partially cross-reactive neutralizing antibodies, recognizing mainly epitopes on EDIII. In the invention, the E80 protein is an N end 80% region of the envelope protein of the Zika virus, is an E protein extracellular section and is responsible for combination with a cell receptor, is a main epitope of the zika virus, and the inventor finds that the E protein secretion is facilitated by cutting a 20% region of the C end of the E protein. The main aim of the invention is to develop a vaccine which can induce the organism to generate target E protein (E80 and EDIII) neutralizing antibodies and is used for preventing the infection of Zika virus.

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

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

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDG AKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNAKNGSISLMCLALGGVLIFLSTAVSA，SEQ ID NO.1。

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

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGK，SEQ ID NO.2。

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

KLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGST，SEQ ID NO.3。

optimization of the Gene sequence encoding the antigenic peptide

In the present invention, there is provided an optimized nucleic acid coding sequence for an antigenic peptide of the invention suitable for expression in a yeast cell.

The present inventors have optimized the DNA sequence encoding the E protein according to the preferred codons without changing its amino acid sequence. However, the inventors found that the optimized sequences obtained solely in terms of codon frequency are not entirely suitable for expression in host cells. The inventor performs secondary optimization, wherein the secondary optimization comprises adjustment and optimization aiming at the GC content of the sequence, so that the region with higher GC content in the original sequence is eliminated; optimizing complex structures (GGGGGG, GGTAAG) such as repeated sequences and cis-acting factors in the original sequence; the rare codons present in the original nucleotide were optimized according to preference (AGG, CGG, GGG, ACG).

Through extensive testing and screening, the present inventors obtained a particularly optimized E protein coding sequence among a number of optimized sequences, the polynucleotide sequences of which are shown below:

atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctggagcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcgaacatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaa cccagggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaatggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagtcgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaacgataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccctgggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgaccatgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacaccggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggtgctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggacgcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaaggccttcgaggccaccgtgcgcggagccaagcgcatggccgtgctgggcgataccgcctgggacttcggaagcgtgggaggagccctgaacagcctgggcaagggcatccaccagatcttcggagccgccttcaagtccctgttcggaggcatgtcgtggttcagccagatcctgatcggcaccctgctgatgtggctgggcctgaacgccaagaatggctccatctcgctgatgtgcctggccctgggaggagtgctgatcttcctgagcaccgccgtgtccgcctaa, SEQ ID NO.4; the sequence codes for the E protein shown in SEQ ID NO. 1.

According to the optimized DNA sequence described above, the DNA sequence encoding E80 is as follows:

atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctggagcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcgaacatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaacccagggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaatggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagtcgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaacgataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccctgggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgaccatgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacaccggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggtgctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggacgcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgtt cggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaag，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 antigen 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, the expression cassette having the following elements in order from 5 '-3': promoters, antigenic peptide genes, and terminators.

The above-described optimized gene sequences of the antigenic peptides can be obtained by conventional methods, such as total artificial synthesis or PCR synthesis, which can be used by those skilled in the art. One preferred synthesis method is an asymmetric PCR method. Primers for PCR can be appropriately selected according to 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 comprising the steps of:

(1) Transforming or transducing a suitable host cell, preferably a yeast or 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 the protein from the culture medium or the cells.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals, preferably commercially available vectors such as pPink. Alpha. HC or 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 an appropriate 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 comprising the above DNA sequences and suitable promoters or control sequences may be used to transform appropriate host cells for expression of the protein of interest. Host cells capable of expressing the antigenic peptides of the invention may beAnd 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 a yeast cell. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. The engineered cells may be those that rapidly utilize methanol (Mut ⁺ ) Or slow utilization of methanol (Mut) ^s )。

Culture of engineering cells and fermentative production of target proteins

After obtaining the engineered cells, the engineered cells may be cultured under appropriate conditions to express the protein encoded by the gene sequence of the present invention. The medium used in the culture may be selected from various conventional media according to the host cell, and the culture is performed under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching 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 present invention are maintained at 28-30 ℃;

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

(c) In the case of Dissolved Oxygen (DO), the DO is controlled to be 20-90%, and the maintenance of dissolved oxygen can be solved by the introduction of oxygen/air mixture;

(d) For the feed, the type of feed preferably includes carbon sources such as glycerin, methanol, glucose, etc., and may be fed alone or in combination.

The engineering cell expressing the target protein may be purified by chromatographic techniques. The chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. 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 high, which affects the binding to the ion exchange medium, the salt concentration is reduced before ion exchange chromatography is performed. The sample can be replaced by dilution, ultrafiltration, dialysis, gel filtration chromatography and other means until the sample is similar to the corresponding ion exchange column equilibrium liquid system, and then the sample is loaded to perform gradient elution of salt concentration or pH.

2. Hydrophobic chromatography

Hydrophobic chromatography media include (but are not limited to): phenyl-Sepharose, butyl-Sepharose, octyle-Sepharose. Sample by adding NaCl, (NH) ₄ ) ₂ SO ₄ And the salt concentration is increased in an equal mode, then the sample is loaded, and the sample is eluted by a method of reducing the salt concentration. The hetero proteins with a large difference in hydrophobicity were removed by hydrophobic chromatography.

3. Gel filtration chromatography

Hydrophobic chromatography media include (but are not limited to): sephacryl, superdex, sephadex. The buffer system is replaced by gel filtration chromatography or further purified.

4. Affinity chromatography

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

Preparation of vaccine compositions

The invention also provides a method of preparing a vaccine composition, in particular comprising the steps of:

The antigenic peptides prepared according to the present invention are mixed with a pharmaceutically acceptable vaccine adjuvant to form a vaccine composition.

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

Compositions and methods of administration

The present invention also provides a composition comprising: (i) Recombinant antigenic peptides prepared by the methods of the invention, and (ii) pharmaceutically or immunologically acceptable excipients or adjuvants. In the present invention, the term "comprising" means that the various ingredients may be applied together or present in the compositions of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

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 compositions of the present invention may be prepared in a variety of conventional dosage forms including, but not limited to: injection, granule, tablet, pill, suppository, capsule, suspension, spray, etc.

(i) Pharmaceutical composition

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

The term "effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective amount for a subject will depend on 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. Thus, it is not useful to pre-specify an accurate effective amount. 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 micrograms/kg to 2 micrograms/kg administered to an individual.

The pharmaceutical composition may also contain 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 agent carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Suitable carriers may be large, slowly metabolizing 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 sufficient 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 can 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. In general, the compositions may be formulated as injectables, either as liquid solutions or suspensions; it can also be made into solid form suitable for formulation into solution or suspension and liquid excipient prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carrier.

(ii) Vaccine composition

The vaccine compositions of the invention may be prophylactic (i.e., to prevent infection) or therapeutic. The vaccine composition comprises an immunological antigen (including a protein of the invention or a self-assembled virus-like particle) and is 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 (e.g. 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 (e.g., a toxoid of a pathogen such as diphtheria, tetanus, cholera, helicobacter pylori, etc.).

Preferred adjuvants that enhance the effect of the immune 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's complete adjuvant (CFA) and Freund's 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 for example WO93/13302 and WO 92-19265; and (7) other substances that act as immunostimulants to enhance the effect 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 specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as other desirable components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prophylaxis, administered to an individual as a single dose or as part of a continuous dose. The amount may depend on the health and physiological condition of the individual being treated, the type 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 this amount will be within a relatively wide range and can be determined by routine experimentation.

Generally, vaccine compositions or immunogenic compositions can be formulated as injectables, such as liquid solutions or suspensions; it can also be made into solid form suitable for formulation into solution or suspension, and liquid excipient prior to 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 a non-human mammal, preferably a human. When used as a vaccine, the virus-like particles of the invention can be administered directly to an individual using known methods. These vaccines are typically administered by the same route of administration as conventional vaccines and/or by a route that mimics pathogen infection.

Routes of administration of the pharmaceutical or vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, intravaginal, oral or other parenteral routes of administration. The routes of administration may be combined, if desired, or adjusted according to the disease condition. The vaccine composition may be administered in a single dose or in 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 the Zika virus infection.

The amount of virus-like particles selected in each vaccine dose is based on the amount that elicits an immunoprotective response without significant side effects. Typically, after infection of the host cells, each dose of vaccine is sufficient to contain about 1 μg to 1000 μg, preferably 1 μg to 100 μg, more preferably 10 μg to 50 μg of protein or VLP. The optimal amount of a particular vaccine can be determined using standard research methods including observing antibody titers and other responses in subjects. Whether an booster dose is required can be determined by monitoring the level of immunity provided by the vaccine. After evaluation of antibody titers in serum, booster dose immunization may be required. The administration of adjuvants and/or immunostimulants may enhance the immune response to the proteins of the invention. The preferred method is to administer the immunogenic composition by injection from a parenteral (subcutaneous or intramuscular) route.

The invention has the main advantages that:

(1) The antigen peptide can be expressed in a large amount in yeast cells, so that the preparation cost is low, and the method 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 quantity in host cells, good stability and suitability for high-density fermentation;

(3) After the antigen peptide EDIII provided by the invention is used for immunizing mice, the immunized mice generate stronger immune response, and combined with an aluminum adjuvant, the antibody induced by the low-dose immunogen ZIKV EDIII is enough to protect AG6 mice from being challenged by the lethal dose of Zika virus;

(4) Compared with the traditional attenuated live vaccine, DNA vaccine and inactivated vaccine, the candidate vaccine is very safe because the candidate vaccine does not have virus nucleic acid.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Materials and methods

1. Cells

Vero cells were cultured in DMEM medium containing 10% bovine serum. Pichia Pink ^TM Yeast strains were purchased from Invitrogen, inc. of America and cultured according to the manufacturer's instructions.

2. Virus (virus)

The ZiKV/SZ-WIV strain used in this study was Asian ZIKV/SZ-WIV strain (GenBank: KU 963796) from the China center for type Collection of microorganisms and viruses (Virus accession number: IVCAS 6.6110).

3. Antibodies to

The antibody of the anti-His tag is a protein intech HRP marked monoclonal antibody, and the anti-ZIKV-E80 antibody is a polyclonal antibody obtained by using E80 protein expressed by insect cells to immunize experimental rabbits in the laboratory.

4. Plasmid construction

Plasmid pZIKV-E was obtained by codon optimization and gene synthesis (SEQ ID NO. 4) based on the E protein coding sequence (SEQ ID NO. 1) of strain Z1106033 (viral nucleotide GenBank: KU312312, amino acid Genbank: ALX 35659) which was popular in south America in Asia 2015 of Zika virus, and further cloning onto vector pUC57 (available from GenScript USA Inc.).

The ED III gene is amplified from a genetically optimized plasmid pZIKV-E by using a primer (ZIKV-ED 3-F: AAGCTGCGCCTGAAGGGC (SEQ ID NO. 7) and ZIKV-ED3-KpnI-R: CAACGGTACCTTAATGGTGATGGTGATGATGGGTGCTTCCCGAGCGGTG (SEQ ID NO. 8)), a fragment with one end being flat and the other end being sticky is generated after single cleavage of the obtained PCR product by KpnI, and is cloned to a StuI and KpnI double-digested pPink alpha-HC (Invitrogen) vector to obtain a plasmid pPink alpha-HC-ZIZI-ED III.

Similarly, the E80 fragment was PCR amplified from plasmid pZIKV-E using primers (ZIKV-E80-F: ATCCGCTGCATCGGCGTGTCGAAT (SEQ ID NO. 9) and ZIKV-E80-KpnI-R: CAACGGTACCTTAATGGTGATGGTGATGATGCTTGCCGATGGTGCTTCC (SEQ ID NO. 10)) and cloned into the pPink α -HC (Invitrogen) vector to give plasmid pPink α -HC-ZIKV-E80.

Pichia 5 transformation and Strain expression level detection

For transformation of Pichia pastoris, plasmids pPink alpha-HC-ZIKV-ED III and pPink alpha-HC-ZIKV-E80 were linearized with EcoNI and then electropositioned into Pichia Pink ^TM Strain 1 (Invitrogen). Transformation of Pichia pastoris and subsequent screening of transformants were performed according to manufacturer's instructions. A small-scale culture expression screening experiment was performed according to the specification. Briefly, transformed yeast single colonies were inoculated into 5ml of BMGY medium, cultured at 30℃for 24hr at 250rpm, and then the supernatant was removed by centrifugation, and the cells were resuspended in 1ml of BMMY medium (containing 1% methanol) and induced at 30℃for 48hr at 250 rpm. After induction, culture supernatant was collected by centrifugation, and Western blotting was performed on the supernatant in an appropriate amount, as described in the previous article.

Preparation of ZIKV-ED III proteins

To prepare the ZIKV-ED III antigen, selected strains were cultured and induced. Culture supernatant was collected by centrifugation, first filtered with a 0.45uM filter membrane, then concentrated by ultrafiltration using a 3KDa ultrafiltration tube, and the concentrated sample was washed from the ultrafiltration tube with Binding buffer and purified using a nickel column. Finally, coomassie brilliant blue staining and Western-blot detection are carried out on the purified protein.

ED III in vitro inhibition Virus infection assay

In order to verify whether the expressed ZIKV-ED III conformation is correct, the inventor carries out ED III in vitro inhibition virus infection test. In short, expressed ZIKV-ED III is diluted 5 times in sequence with 200ug/ml concentration as a starting point to obtain five dilutions, and 100ul of diluted ZIKV-ED III solution is uniformly mixed with 100ul of ZIKV virus (100 PFU)200ul of the mixture was added to one well of a 24-well plate plated with Vero cells, 37 degrees CO ₂ After incubation in the incubator for 1h, the mixture was aspirated, 700ul of DMEM containing 2% FBS with 0.2% agarose was added to each well, left for 15 min at four degrees, and finally the 24 well plate was transferred to 37 degrees CO ₂ After 3-4 days in incubator culture, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

8. Mouse immunity and vaccine specific antibody detection

Purified ED III protein

(Invivogen, U.S.) adjuvants were mixed to make a pilot vaccine, each dose containing 10ug of antigen (ED III) and 500ug of aluminum hydroxide adjuvant. PBS was mixed with adjuvant as a negative control. Balb/c mice (six in each group, 6-8 weeks old) were purchased from Shanghai Laboratory Animal Center (SLAC) and were given intraperitoneal injections of experimental vaccine or PBS negative control at

weeks

0, 2 and 4, and then blood was collected at

weeks

4 and 6 for antibody detection.

The method for determining the ZIKV-ED III specific antibody comprises the following steps: ELISA plates (50 ng/well) were coated with yeast expressed ZIKV-ED III, after each subsequent step of washing the plates three times with PBST to remove non-specific binding, after blocking with 5% nonfat milk powder, 50ul of 1:10000 diluted serum was added to each well, then HRP-conjugated goat anti-mouse IgG (sigma) was added as secondary antibody, TMB color development, 1N phosphate solution stopped, and 0D450 was measured.

9. Virus micro-neutralization assay

The virus micro-neutralization experiment was performed as follows: 24-well culture plate for spreading Vero cells on day before experiment, 10 for each well ⁵ A cell; the next day neutralization experiments were performed by first mixing 100. Mu.l ZIKA virus solution containing 100PFU with 100. Mu.l gradient diluted serum in a 1.5ml EP tube and incubating at 37℃for 1h; the medium in the 24-well plate was then aspirated, washed once with serum-free DMEM, and 200. Mu.l of virus serum mix was added to each well in the 24-well plate, CO at 37 ℃ ₂ After 1h incubation in an incubator, the mixture was aspirated, 700ul of DMEM containing 2% FBS with 0.2% agarose was added to each well, left for 15 min at four degrees, and finally the 24-well plate was transferred to 37 degreesCO ₂ After 3-4 days in incubator culture, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The neutralization titer of the serum samples was defined as the highest serum dilution (PRNT 50) that was able to inhibit 50% of the plaque count compared to the viral-only wells.

10. Toxicity attack protection test for mice

The challenge protection assay was performed on AG6 mice double knocked on type I and type II interferon receptors. The ED III group or PBS group antiserum collected two weeks after three-phase immunity is uniformly mixed with ZIKV/SZ-WIV virus in equal volume, each 100ul of mixture contains 50ul antiserum and 5PFU Zika virus, and then incubated for 1h at 37 ℃. Two groups of 5 week old AG6 mice (5 per group) were intraperitoneally injected with 100 ul/mouse ED III or PBS antiserum-virus mixture, respectively. Mice were weighed 15 days before and after challenge, and survival was observed.

11. Statistics

All statistical analyses were performed using GraghPad Prism version 5. The Kaplan-Meier survival curves were compared using the log-rank test and other experimental data were analyzed using the two-measured student's t-test method. Statistical significance differences were defined as follows: ns, P is more than or equal to 0.05; * P is more than or equal to 0.01 and less than or equal to 0.05; * P <0.01; * P <0.001.

EXAMPLE 1 expression and characterization of ZIKV ED III and E80 in Pichia pastoris

In order to explore recombinant expression of Zika viruses ED III and E80 in Pichia pastoris, the inventors constructed plasmids pPink alpha-HC-ZIKV-ED III and pPink alpha-HC-ZIKV-E80, the plasmid structures of which are shown in FIG. 1. The plasmid pPink alpha-HC-ZIKV-ED III is used for secretory expression of ED III protein, the N-end of the plasmid is fused with alpha-amylase secretion signal peptide, and the C-end of the plasmid is fused with His tag. Similarly, plasmid pPink α -HC-ZIKV-E80 was used to secrete E80 protein with the N-terminus fused to the α -amylase secretion signal peptide and the C-terminus fused to the His tag.

The inventor respectively converts plasmid pPink alpha-HC-ZIKV-ED III and plasmid pPink alpha-HC-ZIKV-E80 into yeast cells, and the obtained recombinant yeast clones detect the expression quantity of ED III and E80 by using a western-blot method, and the yeast cells converted by empty vector pPink alpha-HC are used as negative controls. The result shows that the cloning of the pPink alpha-HC-ZIKV-ED III transformed yeast has very strong His tag resistant monoclonal antibody detection signals (figure 2A), which indicates that the ED III expression level in the yeast is very high; in contrast, the pPink. Alpha. -HC-ZIKV-E80 transformed yeast clone had only a weak signal at the expected molecular weight (FIG. 2B), suggesting that E80 expression levels were low in yeast.

And (3) respectively selecting a No. 4 strain of pPink alpha-HC-ZIKV-ED III and a No. 2 strain of pPink alpha-HC-ZIKV-E80 as high expression strains to perform mass expression purification, and analyzing and identifying the obtained purified products by SDS-PAGE coomassie brilliant blue staining and Western blotting. The protein purified from the pPink alpha-HC-ZIKV-ED III transformed strain 4 exhibited a band of about 13kDa on SDS-PAGE (FIG. 3A), consistent with the molecular weight of the protein calculated from the ED III sequence; positive signals were also detected with the murine anti-His tag antibody and the rabbit anti-ZIKV-E80 antibody at the coomassie brilliant blue stained protein bands, respectively (fig. 3B), indicating that the purified protein was indeed ED iii, with a calculated yield of 4.5mg/l after purification. The protein purified from pPink alpha-HC-ZIKV-E80 transformed strain No. 2 was very few, and there was no clear band (data non show) in SDS-PAGE, probably due to low E80 expression and degradation.

The results show that E80 has poor expression in yeast and is not suitable for research and development of recombinant vaccines. And ED III is correctly and efficiently expressed in yeast, and has great vaccine development potential. Thus, subsequent research has focused on ED III.

Experimental results of expressing ZIKV E80 and ZIKV EDIII by using Drosophila S2 cells show that although the target protein ZIKV E80 cannot be expressed in yeast cells efficiently, the target protein ZIKV E80 can be expressed in Drosophila S2 cells successfully. In contrast, the expression level of ZIKV EDIII in yeast is significantly higher than that in Drosophila S2 cells, and the expression level in yeast is 1.7 times that of Drosophila S2 cells (2.6 mg/l).

EXAMPLE 2 ED III was able to inhibit ZIKV infection in vitro

Mixing purified yeast expressed ED III with ZIKV virus, inoculating Vero cells, and observing plaque number after three days; the control group was set up to incubate Vero cells with BSA and ZIKV virus mixed well. The results show that the control group has no significant difference in plaque number with the reduction of the BSA concentration, which indicates that the BSA cannot inhibit virus infection; in contrast, the number of plaques in the ED III treated group gradually decreased with increasing ED III concentration (FIG. 4A), indicating that ED III was able to dose-dependently inhibit viral infection of cells (FIG. 4B). Calculated from the inhibition curve, a 50% inhibition concentration (IC 50) of ED III of 7.597ug/ml was obtained. This result suggests that yeast expressed ED iii has the correct functional conformation and is able to bind to viral receptors on the cell surface, thereby competitively inhibiting viral binding and entry into the cell.

The experimental results of this example show that the target protein ZIKV EDIII expressed by the yeast according to the invention has inhibitory activity IC on Zika virus infection ₅₀ 7.597ug/ml; while the target protein ZIKV EDIII expressed by Drosophila S2 cells has inhibitory activity IC on Zika virus infection ₅₀ 71.85ug/ml, which is about 10 times different.

Of course, the results of this example also demonstrate that ZIKV EDIII competes with zika virus for entry into cells, and thus has the potential to induce neutralizing antibodies in animals.

EXAMPLE 3 production of high titer neutralizing antibodies by ED III immunized mice

To investigate the immunogenicity of ED III, 2 groups (6 per group) of Balb/c mice were immunized with ED III or PBS, respectively, at week 0, week 2 and week 4, PBS being the negative control group. Serum samples were collected at

weeks

4 and 6, and ELISA assays were performed to detect specific antibody responses using Pichia pastoris expressed ED III as coating antigen. As shown in FIG. 5A, the serum of PBS group mice has a background level of antigen-antibody reaction, while the immune serum of ED III group mice has a remarkable antibody reaction, and the serum of two weeks after three-immunity is higher than the serum of two weeks after two-immunity. ELISA results show that ED III immunized mice produce specific antibodies.

The inventors evaluated the ability of immune sera to inhibit ZIKV infection in vitro by virus neutralization assays. As shown in fig. 5B and 5C, the neutralization titers of PBS group antisera were significantly different from ED iii group antisera; the geometric mean neutralization titer of the ED iii group antisera was 3608. The results show that the ED III vaccine has good immunogenicity and can strongly induce neutralizing antibodies with protective potential.

The experimental result of the embodiment shows that the neutralization activity of the target protein ZIKV EDIII expressed by the yeast to the Zika virus infection is 3608; the neutralization activity of the target protein ZIKV EDIII expressed by Drosophila S2 cells on the infection of the Zika virus is 1633.8, and the difference between the two is about 3 times.

Example 4.Ed iii immunized mouse serum has antiviral function in vivo.

To evaluate the protective effect of immune serum in mice, type I and type II interferon receptor deficient AG6 mice sensitive to wild type ZIKV were selected for challenge protection testing. Each AG6 mouse was intraperitoneally injected with ED III or PBS antiserum in combination with ZIKV for 14 days, and the change in body weight and survival rate of the mice were recorded. As shown in fig. 6, PBS group mice began to lose weight on the fifth day after challenge, and died all over the 9 th-10 th day; in contrast, ED III antiserum treated mice have a gentle rise in body weight after challenge, with 100% survival rate. The data indicate that ED III vaccine group serum has a protective effect in mice and can prevent the attack of the Zika virus with lethal dose.

Comparative example 1 expression of the proteins ZIKV E80 and ZIKV EDIII in Drosophila S2 cells

Drosophila Schneider 2 (S2) cells were purchased from Invitrogen and 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 incubated in an incubator at 28 ℃.

Drosophila cell expression vector pMT/BiP/V5-HisA, screening plasmid pCoBlast and calcium phosphate transfection kit were purchased from Invitrogen corporation. The plasmid pZIKV-E was obtained by codon optimization and gene synthesis from the E protein coding sequence (SEQ ID NO. 1) of strain Z1106033 (viral nucleotide GenBank: KU312312, amino acid Genbank: ALX 35659) popular in south America in Asia 2015 of Zika virus, and further cloning onto a vector pUC57 (purchased from GenScript USA Inc.). After pZIKV-E is used as a template and amplified by a specific primer PCR, the two ends of the template are provided with Bgl II and Xba I enzyme cutting sites, and the template is connected to an insect expression vector pMT/Bip/V5-His A (containing a His tag and being beneficial to detection and purification of target proteins) containing Bgl II and Xba I enzyme cutting sites, so that recombinant plasmids pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII carrying the N end 80% region (ZIKV E80) of the Zika virus envelope protein and the target gene fragment of the envelope protein region III (ZIKV EDIII) are obtained.

The constructed recombinant plasmids pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII (shown in figure 1) are transiently transfected into Drosophila cells, and the target protein is detected by performing western blot detection on culture medium supernatant after chromium chloride induction. Then co-transferring the recombinant plasmid and pCoBlast screening plasmid, screening cells of a stable line, carrying out induced expression, obtaining cell supernatant, purifying, wherein SDS-PAGE shows that the size of ZIKV E80 protein is 54KD (shown in figure 2A), using a mouse anti-His-tag antibody as a primary antibody, and detecting that the size of ZIKV E80 (shown in figure 2B) is consistent with the result of SDS-PAGE by western blot. Similarly, ZIKV EDIII has a size of 15KD and is consistent with the size of a band detected by a western blot. The result shows that the target protein E80 and the EDIII are expressed, and the expression yields of the ZIKV E80 and the ZIKV EDIII antigen peptide are calculated to reach 10mg/l and 2.6mg/l after purification.

Conclusion(s)

The inventor obtains a stable transfer cell line by adopting a yeast cell system and expresses a truncated envelope protein EDIII of the Zika virus. The target protein ZIKV EDIII obtained by the invention has a good inhibition effect in the experiment of inhibiting the Zika virus infected cells. After the third immunization of the BALB/c mice, the immunized mice developed a stronger immune response, both in terms of serum antibody titer and specific T cell response. Most importantly, in combination with aluminum adjuvant, the antibody induced with the low dose of the immunogen ZIKV EDIII was sufficient to protect AG6 mice from challenge with lethal doses of zika virus.

In the aspect of preparing target proteins, the applicant finds that the expression is carried out by adopting different host cells, and under the condition that the gene sequences are identical, the expression quantity of antigen peptides is greatly different and the protein activity is greatly different, for example, the ZIKV EDIII antigen peptides of the invention are expressed by adopting yeast cellsThe expression level of (2) can reach 4.5mg/l, and the expression level of Drosophila S2 cells is only 2.6mg/l. Compared with Drosophila S2 cells, the expression level of yeast is improved by more than 70%. In terms of activity, the ZIKV EDIII antigen peptides expressed by the yeast of the invention have inhibitory activity (IC) on Zika virus infection ₅₀ Inhibitory Activity of ZIKV EDIII antigen peptides on Drosophila S2 cell surface against Ziv virus Infection (IC) = 7.597 ug/ml) ₅₀ Compared with 71.85 ug/ml), the inhibiting activity of ZIKV EDIII antigen peptide expressed by yeast on Ziv Ka virus is improved by about 10 times, more importantly, the ZIKV EDIII antigen peptide expressed by yeast cells has unexpected excellent technical effect on the neutralization level, the ZIKV EDIII group serum expressed by yeast has stronger neutralization capability and PRNT ₅₀ Reach 3608, and the ZIKV EDIII group serum neutralization capacity PRNT expressed by Drosophila S2 cells ₅₀ 1633.8. Compared with ZIKV EDIII antigen peptide expressed by Drosophila S2 cells, the capability of the ZIKV EDIII antigen peptide expressed by yeast to induce neutralizing antibodies is improved by 120%. Yeast-expressed ZIKV EDIII antigenic peptides are significantly advantageous as candidate vaccines for Zika virus in a comprehensive view of expression yield, antigenic peptide activity and level of neutralization of induced antibodies.

Compared with the traditional attenuated live vaccine, DNA vaccine and inactivated vaccine, the candidate vaccine is very safe because the candidate vaccine does not have virus nucleic acid. In addition, the purification is convenient, no complex technology is needed, the operation is simple, and the method has relatively large development potential.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp

370 375 380

Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp

385 390 395 400

His Arg Ser Gly Ser Thr Ile Gly Lys

405

<210> 3

<211> 110

<212> PRT

<213> Zika virus

<400> 3

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> 18

<212> DNA

<213> artificial sequence

<400> 7

aagctgcgcc tgaagggc 18

<210> 8

<211> 49

<212> DNA

<213> artificial sequence

<400> 8

caacggtacc ttaatggtga tggtgatgat gggtgcttcc cgagcggtg 49

<210> 9

<211> 24

<212> DNA

<213> artificial sequence

<400> 9

atccgctgca tcggcgtgtc gaat 24

<210> 10

<211> 49

<212> DNA

<213> artificial sequence

<400> 10

caacggtacc ttaatggtga tggtgatgat gcttgccgat ggtgcttcc 49

Claims

1. An antigenic peptide, wherein said antigenic peptide is derived from an envelope protein of a zika virus, and

the amino acid sequence of the antigen peptide is shown as SEQ ID NO.3, and the antigen peptide is recombinant protein expressed by yeast cells.

2. A host cell expressing the antigenic peptide of claim 1, wherein the host cell is a yeast.

3. A pharmaceutical composition comprising the antigenic peptide of claim 1 or the host cell of claim 2, and a pharmaceutically acceptable carrier and/or adjuvant.

4. A pharmaceutical composition according to claim 3, wherein the composition is a vaccine.

5. A vaccine composition comprising the antigenic peptide of claim 1 or the host cell of claim 2, together with an immunologically acceptable carrier and/or adjuvant.

6. The vaccine composition of claim 5, further comprising an adjuvant.

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

8. The use of claim 7, wherein said disease associated with the zika virus comprises: zika virus infection, green's barker syndrome, and microcephaly.

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

(i) Culturing the host cell of claim 2 under suitable conditions to express the antigenic peptide of claim 1;

(ii) Purifying the antigenic peptide.