CN117126253A

CN117126253A - HSV immunogenic recombinant protein, preparation method and application thereof, and vaccine prepared by using same

Info

Publication number: CN117126253A
Application number: CN202310439228.XA
Authority: CN
Inventors: 张岭; 张元杰; 邓家荔; 郝怡环; 罗士强; 潘勇昭; 洪坤学; 陈健平; 刘勇
Original assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Current assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Priority date: 2022-05-27
Filing date: 2023-04-23
Publication date: 2023-11-28
Anticipated expiration: 2043-04-23
Also published as: CN117122676A; CN117126253B

Abstract

The application provides an HSV immunogenicity recombinant protein, which comprises gB protein extracellular domain or functional fragment thereof, provides highly conserved epitope directly related to HSV virus invasion cells, can effectively stimulate the production of specific neutralizing antibodies and cytokines, and stimulates higher level humoral immunity and cellular immunity; the vaccine prepared by the recombinant protein can generate good immune effect against various HSV strains, thereby providing broad-spectrum and high-efficiency protection and effectively preventing HSV infection.

Description

HSV immunogenic recombinant protein, preparation method and application thereof, and vaccine prepared by using same

Technical Field

The application belongs to the field of biomedical engineering, and in particular relates to an HSV immunogenicity recombinant protein, a preparation method thereof and application thereof in preparing vaccines.

Background

Herpes simplex virus (herpes simplex virus, HSV) is one of the most common human infectious viruses, widely found in nature, and causes lesions primarily by infection of the host through skin, mucous membranes and nervous tissue. HSV can be divided into two serotypes of HSV-1 and HSV-2, and after HSV-1 infects human body, it mainly causes herpes labialis, pharyngitis and keratitis, and even can cause severe diseases such as sporadic encephalitis; HSV-2 mainly causes genital herpes, neonatal herpes and the like through damaged skin and mucosal infection.

HSV is a double-stranded DNA virus, is spherical and consists of a core, a capsid, a coating and an envelope, and has a virus particle diameter of about 150-200 nm. The surface of the envelope has gB, gC, gD, gE, gF, gG, gH, gI, gJ, gK, gL, gM and other 12 glycoproteins, of which gB, gD, gH, gK and gL are necessary for infecting cells. The optional viral glycoprotein gC plays a major role in the initial association of the virus with the cellular receptor, heparin sulfate portion of the cellular proteoglycan, whereas both gC and gE/gI proteins are involved in immune escape of HSV virus.

The first step of HSV infection is membrane fusion, and related proteins are gB, gD and gH/gL, which play an important role in HSV invading cells, and the HSV fusion process mediated by the proteins is as follows: (1) the gD protein binds to the receptor and undergoes a conformational change; (2) The conformationally altered gD facilitates conversion of gH/gL to a form capable of interacting with gB, thereby activating gB; (3) The activated gB must, together with the gH/gL, promote the attachment of the virus to the cell membrane. Of these, only gB is believed to have a fusion-promoting effect, which undergoes conformational changes during HSV fusion, which induces the production of specific neutralizing antibodies, and also the production of cd4+ and cd8+ T cell immunity.

The gB protein is highly conserved in all herpesvirus families, is also an important target for humoral immunity and cellular immunity, and therefore becomes an important gene in the development of HSV vaccines. The gB protein is encoded by the UL27 gene, has a total length of 904 amino acids, and has a structure shown in FIG. 1, and a Signal Peptide (SP), an extracellular domain (extracellular domain, ECD), a membrane proximal region (membrane proximal region, MPR), a transmembrane domain (transmembrane domain, TM) and a cytoplasmic region (cytoplasmic domain, CTD) are arranged from the N-terminal to the C-terminal. The Fusion ability of the gB protein is derived from the extracellular domain, which contains I, II, III, IV, V five regions, where I and II are Fusion (Fusion) domains (Vollmer, b., et al The prefusion structure of herpes simplex virus glycoprotein b.sci Adv,2020.6 (39)). The natural gB protein is in a trimer structure, and promotes fusion of a virus membrane and a cell membrane by combining with a cell surface heparan sulfate protein, so that the virus is mediated to enter the cell. Thus, immune protection against the gB protein is the first step in preventing infection.

Disclosure of Invention

In order to solve the technical problems, the application provides an HSV immunogenicity recombinant protein, a preparation method and application thereof, and a vaccine prepared by using the same for preventing HSV infection.

The technical scheme of the application is as follows:

in one aspect the application provides an HSV immunogenic recombinant protein comprising, or being at least 90% identical in sequence to, the extracellular domain of a gB protein of at least one of HSV-1 and HSV-2, or a functional fragment thereof; wherein the functional fragment comprises at least domains I and II of the extracellular domain of the gB protein of at least one of HSV-1 and HSV-2.

Further, the recombinant protein comprises the extracellular domain of the gB protein of HSV-1 or a functional fragment thereof and the extracellular domain of the gB protein of HSV-2 or a functional fragment thereof.

Further, the C-terminal of the gB protein ectodomain or the functional fragment thereof of the HSV-1 is directly or indirectly connected with the N-terminal of the gB protein ectodomain or the functional fragment thereof of the HSV-2, or the C-terminal of the gB protein ectodomain or the functional fragment thereof of the HSV-2 is directly or indirectly connected with the N-terminal of the gB protein ectodomain or the functional fragment thereof of the HSV-1.

For example, in one embodiment, the C-terminus of the extracellular domain of the gB protein of HSV-1 is directly or indirectly linked to the N-terminus of the extracellular domain of the gB protein of HSV-2. In one embodiment, the C-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-1 is directly or indirectly linked to the N-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-2. In one embodiment, the C-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-1 is directly or indirectly linked to the N-terminus of the extracellular domain of the gB protein of HSV-2. In one embodiment, the C-terminus of the gB protein extracellular domain of HSV-1 is directly or indirectly linked to the N-terminus of a functional fragment of the gB protein extracellular domain of HSV-2.

In another embodiment, the C-terminus of the extracellular domain of the gB protein of HSV-2 is directly or indirectly linked to the N-terminus of the extracellular domain of the gB protein of HSV-1. In another embodiment, the C-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-2 is directly or indirectly linked to the N-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-1. In another embodiment, the C-terminus of the functional fragment of the extracellular domain of the gB protein of HSV-2 is directly or indirectly linked to the N-terminus of the extracellular domain of the gB protein of HSV-1. In another embodiment, the C-terminus of the extracellular domain of the gB protein of HSV-2 is directly or indirectly linked to the N-terminus of a functional fragment of the extracellular domain of the gB protein of HSV-1.

Further, the extracellular domain of the gB protein or a functional fragment thereof is mutated by at least one amino acid.

Further, the amino acid sequence of the recombinant protein is at least 90% identical to any one of SEQ ID NOS.1-7.

The application also provides a preparation method of the HSV immunogenicity recombinant protein, which comprises the following steps:

s1: synthesizing a DNA sequence corresponding to the recombinant protein, and cloning the DNA sequence onto a plasmid vector;

s2: transfecting a host cell with the plasmid vector and expressing the host cell, wherein the host cell is a eukaryotic cell;

s3: purifying the culture product to obtain the recombinant protein.

Further, the plasmid vector is pRK1.6 vector or pcDNA3.1 vector.

Further, in step S2, the method of transfecting the host cell is as follows:

diluting the transfection system by 10 times by using a culture medium to obtain an incubation system;

adding the PEI mixture into the DNA mixture according to a proportion, standing and incubating for 1-10 min (for example, 5 min);

the PEI-DNA mixture was mixed with the host cells and cultured.

Further, the host cell is an Expi CHO-S cell.

Further, in step S2, the medium used for amplification is EmCD CHO-S203 medium.

Further, the amount of DNA used in transfecting the host cell with the plasmid vector is 1. Mu.g/mL, the volume ratio of PEI to DNA solution is 2:1 to 6:1, specifically may be 2:1, 3:1 or 4:1, preferably 6:1.

Further, the environmental conditions of the culture are 37+/-2 ℃ and 8% CO ₂ A humid incubator at 100 to 150rpm (e.g., 120 rpm).

Further, the culturing further comprises the following steps:

after culturing for 24 hours, the temperature is reduced to 32+/-2 ℃ for continuous culturing for 3-8 days.

Further, the culturing further comprises the following steps:

medium feeding was performed at 3 to 8% (e.g., 5%) by volume on days 1, 3, and 5 of the culture, and the feeding component was Advanced feed1 or Em-S1F from Sigma Co.

Further, in step S3, the culture product is purified by metal chelate chromatography, eluting with imidazole-PBS system.

Further, the method comprises the following steps before purification by metal chelate chromatography:

centrifuging and filtering the culture product, and adding imidazole to the filtered fermentation broth to obtain a final concentration of imidazole in the fermentation broth of 5-15 mM (e.g., 10 mM).

Further, the imidazole-PBS system comprises a washing liquid for washing impurities attached to the chromatographic column after chromatography, and an eluent for eluting the proteins adsorbed on the chromatographic column after washing, wherein the washing liquid is a PBS buffer solution containing 10-50 mM (e.g. 30 mM) imidazole, and the eluent is a PBS buffer solution containing 200-1000 mM (e.g. 500 mM) imidazole.

Further, the method also comprises the following steps after elution:

concentrating and centrifuging the eluted protein, adding 10-20 times of PBS buffer solution, uniformly mixing, centrifuging, repeating for three times, replacing the solvent of the protein solution, and diluting.

Further, in step S3, the pH value of all the reaction systems is controlled to be 7-8, for example, 7.4.

The application also provides application of the recombinant protein in preparing a vaccine for preventing HSV infection.

Further, the recombinant protein is used in a single vaccine at a dose of 20 to 80 μg (e.g., 50 μg).

Further, in the vaccine, the recombinant protein is used in combination with at least one immunoadjuvant selected from at least one of aluminum adjuvants, squalene, tocopherol, MPL, LPA, cpG, poly (I: C), and QS-21.

Further, the human dose of the recombinant protein is 40-160 μg, dispersed in two doses of the same vaccine administered at 14-28 days intervals.

In a fourth aspect the present application provides a vaccine for preventing HSV infection comprising the recombinant protein described above; the vaccine provides protection against at least one of the two serotypes HSV-1 and HSV-2.

Further, the single vaccine contains 20-80 mug of the recombinant protein.

Further, the single dose vaccine contains 50 mug of the recombinant protein.

Further, an immunoadjuvant selected from at least one of aluminum adjuvants, squalene, tocopherol, MPL, LPA, cpG, poly (I: C), and QS-21 is also contained.

Further, the immunoadjuvants are MPL and QS-21.

Further, the single vaccine contains MPL 10-100 μg (e.g., 50 μg) and QS-21 10-100 μg (e.g., 50 μg).

Further, the immunoadjuvant is CpG.

Further, the single dose vaccine contains CpG 2.5-5 mg (e.g., 3.155 mg).

Further, the immunoadjuvant is CpG and aluminum hydroxide.

Further, the single vaccine contains CpG 2.5-5 mg (e.g., 3.155 mg) and aluminum hydroxide 0.1-1 mg (e.g., 0.5 mg).

Further, the vaccine comprises a first dose and a second dose of the same composition administered at 14-28 days (e.g., 21 days) intervals.

The beneficial effects of the application are as follows: the application provides an HSV immunogenicity recombinant protein, which comprises gB protein extracellular domain or functional fragment thereof, provides highly conserved epitope directly related to HSV virus invasion cells, can effectively stimulate the production of specific neutralizing antibodies and cytokines, and stimulates higher level humoral immunity and cellular immunity; the vaccine prepared by the recombinant protein can generate good immune effect against various HSV strains, thereby providing broad-spectrum and high-efficiency protection and effectively preventing HSV infection.

Drawings

FIG. 1 is a schematic diagram of the structure of HSV virus gB protein;

FIG. 2 is an SDS-PAGE electrophoresis of different recombinant plasmids, wherein: (a) Lanes 1-8 in (a) are pcDNA3.1-gB1-P, pcDNA3.1-gB1-fusion, pRK1.6-gB1-WT, pcDNA3.1-gB1-WT, pRK1.6-gB2-fusion, pcDNA3.1-gB2-fusion, pRK1.6-gB2-WT, pcDNA3.1-gB2-WT; (b) Lanes 1-3 (from left to right) in the sequence pcDNA3.1-gB1-gB2-fusion, pcDNA3.1-gB2-P, pcDNA3.1-gB2-WT;

FIG. 3 is an agarose gel electrophoresis diagram of different recombinant plasmids, wherein: (a) From left to right, pRK1.6-gB1-P, pcDNA3.1-gB1-P, pRK1.6-gB1-fusion, pcDNA3.1-gB1-fusion, pRK1.6-gB1-WT, pcDNA3.1-gB1-WT, pRK1.6-gB2-fusion, pcDNA3.1-gB2-fusion, pRK1.6-gB2-WT, pcDNA3.1-gB2-WT, pRK1.6-gB2-P; (b) The lanes from left to right are pcDNA3.1-gB2-P, pcDNA3.1-gB2-WT, pcDNA3.1-gB1-gB2-fusion, pRK1.6-gB2-WT;

FIG. 4 shows the transient transformation test results of pcDNA3.1-gB1-P plasmid, wherein (a), (b) and (c) are SDS-PAGE electrophoresis of groups 1 to 3, respectively;

FIG. 5 shows the results of transient experiments on pcDNA3.1-gB1-fusion plasmids, wherein (a), (b) and (c) are SDS-PAGE patterns of groups 1 to 2, 3 to 4 and 5, respectively;

FIG. 6 shows the transient experimental results of pcDNA3.1-gB1-WT, pcDNA3.1-gB2-fusion and pcDNA3.1-gB1-gB2-fusion plasmids;

FIG. 7 is a graph showing CD4 after immunization with different vaccines ⁺ Different cytokine detection results in T cells;

FIG. 8 is a comparison of cytokine production after immunization with different vaccines;

FIG. 9 shows the results of different vaccines against gB1-P antigen for the induction of specific IgG antibodies;

FIG. 10 is the results of different vaccines against gB1-fusion antigen for the induction of specific IgG antibodies;

FIG. 11 shows the results of specific IgA antibody induction by different vaccines;

FIG. 12 shows the results of neutralization experiments of serum HSV-1 strains after immunization with different vaccines;

FIG. 13 shows the results of a serum HSV-2 strain neutralization assay after immunization with Gr 3;

FIG. 14 is CD4 ⁺ Intracellular cytokine detection of T cells;

FIG. 15 is vaccine-induced serum-specific IgG antibody titers (GMT, geometric mean);

figure 16 is vaccine induced serum-specific neutralizing antibody titers.

Detailed Description

The application is described in further detail below with reference to the drawings and examples. It should be noted that the nomenclature of recombinant proteins is only used for convenience, wherein "gB1" represents that the protein is derived from HSV-1 (wild type full length sequence: AAF 04615.1), "gB2" represents that the protein is derived from HSV-2 (wild type full length sequence: AAA 66440.1), for example, gB1-WT represents that the protein is directly truncated from the extracellular domain of wild type HSV-1gB protein (i.e., 31-730 of wild type gB protein, 1-30 of gB protein is signal peptide), gB2-WT represents that the protein is directly truncated from the extracellular domain of wild type HSV-2 (i.e., 23-771 of wild type gB protein, 1-22 of gB protein is signal peptide), gB1-P represents that the protein is taken from the extracellular domain of HSV-1 and proline substitution occurs at 516, gB2-P represents that the protein is taken from the extracellular domain of HSV-1gB protein, and Fusion domain of HSV-2 (I-2 is the Fusion domain of HSV-1) and Fusion domain of Fusion 1-52 is directly truncated to 2 (I-Fusion domain of wild type HSV-1, 2-Fusion 1).

In the present application, the term "direct connection" or "indirect connection" generally refers to two different connection modes, i.e., direct connection and indirect connection of two sequences, wherein the direct connection refers to the connection between two sequences without any artificial addition of other sequences, such as flexible Linker, rigid Linker or cleavable Linker. The indirect connection means that the two-segment sequence is connected by means of artificial addition of connecting sequences, such as flexible Linker, rigid Linker or shearable Linker. In certain embodiments, the flexible Linker may comprise one or more GSGSG, GGGSS, GGGS, etc., amino acid sequences.

In the present application, pRK1.6 vector means a vector obtained by adding a TM element downstream of CMV promoter of pcDNA3.1 vector and a WPRE element downstream of MCS, wherein the TM element means a triplet leader sequence of human adenovirus Major Late Promoter (MLP) mRNA and the WPRE element is a woodchuck hepatitis virus posttranscriptional regulatory element.

EXAMPLE 1 Synthesis of fusion genes

The gene sequences were designed and codon optimized according to the antigen proteins in table 1, and the corresponding fusion genes were synthesized artificially.

TABLE 1 antigenic gene sequences

EXAMPLE 2 construction of recombinant plasmid vector

The first 6 of the above antigen genes were cloned and ligated to pRK1.6 and pcDNA3.1 vectors, and the gB1-gB2-fusion gene was ligated to pcDNA3.1 vector to construct recombinant plasmid vectors expressing seven antigen proteins, respectively, and transfected into CHO cells as follows:

expi CHO-S cells (Thermo) were cultured in EmCD CHO-S203 (Eminence/L20301) supplemented with 6mM L-glutamine (sigma/G5146) at a DNA dose of 1. Mu.g/ml, transfection reagent PEI (Polysciences/24765-1) to DNA ratio of 6:1; compounding with the above-mentioned L-glutamine-containing mediumPreparation of transfection incubation System (volume 10% of transfection volume), addition of PEI mixture to DNA mixture, standing incubation for 5min, culture at 37deg.C, 8% CO ₂ Shake flask culture in a humid incubator at 120 rpm; after 24h of transfection, the temperature is reduced to 32 ℃ for culture, advanced Feed1 (sigma/24368-1L) is added according to the volume ratio of 5% in days 1, 3 and 5 after transfection for feeding, the sugar concentration is kept to be not lower than 2g/L, the culture is stopped on day 6 after transfection, and the supernatant is taken for SDS-PAGE detection. As a result, clear bands can be seen at the corresponding size positions, as shown in FIG. 2, indicating that the recombinant plasmid vector carrying the target gene has been successfully transferred into CHO cells.

Example 3 recombinant plasmid vector amplification and characterization

The glycerol bacteria transformed with the plasmid containing the objective gene were transferred into 150mL LB liquid medium (Amp final concentration 100. Mu.g/mL), shake cultured overnight at 37℃and 2000rpm, plasmid extraction was performed using endotoxin-free plasmid large extraction kit, and the extract was subjected to plasmid concentration determination and cleavage identification, wherein pcDNA3.1 plasmid was double-digested with NotI and EcoRI, pRK1.6 plasmid was single-digested with NotI, and cleavage was performed at 37℃for 2 hours, and the specific reaction system was as shown in Table 2.

TABLE 2 cleavage reaction System

(1) The concentration was measured after plasmid amplification, and the results are shown in Table 3, and it was found that each plasmid could obtain a higher amplification concentration.

TABLE 3 plasmid concentration measurement results

(2) The cleavage results were detected by 1% agarose gel electrophoresis, and clear bands were visible at the corresponding size positions as shown in FIG. 3.

(3) All plasmids were sequenced and, as identified, all gB plasmids were completely correct in the gene sequence of interest.

In conclusion, it is known that the recombinant plasmid vector carrying the target gene has been successfully transferred into host cells and amplified.

EXAMPLE 4 antigen protein purification

The CHO cell broth obtained in example 2 was centrifuged at 4000rpm for 10min, filtered with a 0.22 μm PVDF filter (Millipore) and the supernatant was retained; 10ml of a metal chelating chromatography purification packing (Zhongkesen bright) was filled into an XK16/20 empty chromatography column (cytova), then connected to an AKTA pure purifier (cytova), and the chromatography column was equilibrated with PBS buffer at pH7.4 to a baseline equilibrium of conductivity and absorbance; imidazole was added to 600ml of the filtered broth to a concentration of 10mM and was fed by a purifier pump. After the sample injection, the target protein was washed to baseline equilibrium with 30mM imidazole-containing PBS buffer pH7.4, eluted with 500mM imidazole-containing PBS buffer pH7.4, the eluates were pooled, transferred to a 30kDa MWCO ultrafiltration tube (Millipore), centrifuged at 4000rpm, concentrated to a liquid volume of about 1ml, and then 15ml of pH7.4 PBS buffer was added to resuspend and centrifuged, repeated three times, and the solution system of the protein solution was replaced with PBS and diluted. SDS-PAGE gel analysis and WB analysis were performed on the obtained protein solution, and protein concentration was determined using BCA method.

The result shows that the highest concentration of the purified protein can reach 3.45mg/ml, the yield can reach 50%, obvious bands are visible at the corresponding size position in the gel, and few and unobvious bands are generated, so that the purification method can obtain the target protein product with high yield and high purity.

EXAMPLE 5 antigenic protein mass analysis

(1) Particle size detection

The particle size of the target protein obtained by extraction and purification in example 4 was measured and repeatedly detected three times by a malvern particle sizer, and the results show that the particle size of each protein product is smaller than 30nm and the distribution degree PDI is smaller than 0.2, which indicates that the purified protein has smaller particle size, good uniformity and difficult aggregation.

(2) Stability detection

The results of high-temperature acceleration test and long-term test on the target protein gB1-gB2-fusion show that the TM value (the temperature at which the protein starts to aggregate) is very close to that of the monoclonal antibody lgG (control), and the temperature is higher than 70 ℃, which shows that the purified protein has stable properties and is convenient to store and transport.

Example 6 transient condition optimization experiment

6.1g B1-P plasmid transient condition optimization experiment

A transient transformation experiment of pcDNA3.1-gB1-fusion plasmid was performed according to the transfection method provided in example 2, and PEI/DNA mixed system was prepared according to the conditions in Table 4, respectively, and fed during the cultivation, during which the sugar concentration was monitored daily and fed to 5g/L, and the sugar content was maintained at not less than 2g/L; samples were collected from day 5 after transfection (supernatant+cells) until the activity reached about 70%, and SDS-PAGE was performed on the products.

TABLE 4 transient protocol for the gB1-P vector

The experimental results are shown in figure 4, obvious bands appear in each group, wherein 1-3 groups have equivalent definition, which indicates that the expression amounts of several PEI/DNA ratios are equivalent and better expression effect can be obtained; further, it is clear from the figure that the prolonged culture time does not increase the expression level, but rather causes a significant impurity band, which is unfavorable for the subsequent purification, and therefore, it is preferable to terminate the culture after 5 to 7 days of collection after the transient transformation.

6.2g B1-fusion plasmid transient condition optimization experiment

A transient transformation experiment of pcDNA3.1-gB1-fusion plasmid was performed according to the transfection method provided in example 2, and PEI/DNA mixed system was prepared according to the conditions in Table 5, respectively, and fed during the cultivation, during which the sugar concentration was monitored daily and fed to 5g/L, and the sugar content was maintained at not less than 2g/L; samples were collected from day 5 after transfection (supernatant+cells) until the activity reached about 70%, and SDS-PAGE was performed on the products.

TABLE 5 scheme for transient transformation of gB1-fusion vectors

The experimental results are shown in FIG. 5, obvious bands appear in each group, wherein 1-3 groups have equivalent definition, which indicates that the expression amounts of several PEI/DNA ratios are equivalent and better expression effect can be obtained; the 3-5 groups compare different feed components and feed amounts, and the results show that the influence of the different feed components on the expression amount is not greatly different.

In summary, the suitable ratio of PEI/DNA is wider, but for large-scale preparation, the ratio of 6:1 is preferable, so that the use amount of DNA can be effectively saved while the expression effect is ensured; meanwhile, both feed1 and Em-S1F are suitable as components of the feed, but feed1 is lower in cost than Em-S1F and higher in activity on the day of liquid collection, and feed1 is preferred for mass production.

FIG. 6 is a SDS-PAGE electrophoresis of gB1-WT, gB2-fusion and gB1-gB2-fusion obtained under optimized transient conditions.

EXAMPLE 7 preparation of antigen working fluid

gB1-P (original concentration 3.45 mg/ml) was used as antigen, and 29. Mu.l of antigen stock solution was mixed with 971. Mu.l of antigen buffer (1 XPBS buffer) to obtain gB1-P antigen working solution.

Example 8 preparation of antigen working fluid

gB1-fusion (original concentration 1.65 mg/ml) was used as antigen, and 24.2. Mu.l of antigen stock solution was mixed with 375.8. Mu.l of antigen buffer (1 XPBS buffer) to obtain gB1-fusion antigen working solution.

Example 9 preparation of adjuvant working fluid

Mu.l of CpG (concentration 32.42 mg/ml) was mixed with 245. Mu.l of 154mM NaCl solution to obtain a CpG adjuvant working solution.

EXAMPLE 10 preparation of adjuvant working fluid

60 μl of Al (OH) was taken ₃ (available from Croda Denmark, concentration 2%, aluminum content 10mg/ml, CAS: 21645-51-2), added to 430. Mu.l of 154mM NaCl solution, and mixed well for use; adding 110 μl CpG into the above solution; adsorbing the obtained solution on a shaking table at 20rpm for 30min to obtain CpG+Al (OH) ₃ Adjuvant working fluid.

EXAMPLE 11 preparation of adjuvant working fluid

DOPC (dioleoyl phosphatidylcholine), cholesterol and MPL were dissolved in ethanol, a lipid film was formed by evaporating the solvent under vacuum, phosphate buffer made up of sodium chloride, anhydrous disodium hydrogen phosphate and potassium dihydrogen phosphate was added and the mixture was pre-homogenized, followed by microfluidization (20 cycles) at 15,000psi, and the resulting liposomes were aseptically filtered through a 0.22 μm film to give an AS01 adjuvant working solution containing 50. Mu.g MPL, 50. Mu.g QS-21, 1mg DOPC and 0.25mg cholesterol per 0.5ml AS01 adjuvant working solution.

EXAMPLE 12 preparation of vaccine working fluid

Mu.l of the antigen working solution provided in example 7 was mixed with 300. Mu.l of the adjuvant working solution provided in example 10 to obtain Gr1 vaccine working solution.

EXAMPLE 13 preparation of vaccine working fluid

Mu.l of the antigen working solution provided in example 7 was mixed with 300. Mu.l of the adjuvant working solution provided in example 9 to obtain Gr2 vaccine working solution.

EXAMPLE 14 preparation of vaccine working fluid

Mu.l of the antigen working solution provided in example 7 was mixed with 300. Mu.l of the adjuvant working solution provided in example 11 to obtain Gr3 vaccine working solution.

EXAMPLE 15 preparation of vaccine working fluid

Mu.l of the antigen working solution provided in example 8 was mixed with 300. Mu.l of the adjuvant working solution provided in example 10 to obtain Gr4 vaccine working solution.

EXAMPLE 16 HSV vaccine immunization efficacy test with different adjuvants

20C 57BL/6N mice (female, 6-8 weeks old, purchased from Beijing Vitrending laboratory animal technologies Co., ltd.) were randomly divided into 4 groups of 5 mice each, and were subjected to group immunization in the manner shown in Table 6, and each index was observed and measured.

TABLE 6 grouping experiment design cases

Group of	Vaccine working fluid	Immunization dose	Immunization pathway and volume	Immunization program
					1	Gr1	1/10HD	Intradermal 0.1ml	2 doses, interval 21d
2	Gr2	1/10HD	Intradermal 0.1ml	2 doses, interval 21d
					3	Gr3	1/10HD	Intradermal 0.1ml	2 doses, interval 21d
4	Gr4	1/10HD	Intradermal 0.1ml	2 doses, interval 21d

16.1 general clinical observations and death Condition

During animal immunization, the animal status and the administration site were observed on the day of immunization.

16.2 blood sample collection and processing

Blood collection is carried out on day 0, day 21, day 35 and day 49 of immunization respectively, and the blood collection method is that blood collection is carried out on the posterior ocular venous plexus, and the blood collection amount is 300-500 mu l each time; after blood collection, the sample was not anticoagulated, placed at 37℃for 1h, then placed at 4℃for 0.5h, centrifuged at 8000rpm for 10min, serum was collected, inactivated in a 56℃water bath for 30min, and stored at-20 ℃.

16.3 antigen-specific IgG antibody detection

The antigen-specific IgG antibodies were assayed by ELISA, as follows:

(1) Mice were bled 14 days after the last immunization (35D), gB1-P antigen was diluted to 3. Mu.g/ml with CB coating solution (pH 7.4.+ -. 0.1) (or gB1-fusion antigen was diluted to 5. Mu.g/ml), 100. Mu.l each well was added to the 96-well plate, coated overnight at 2-8 ℃ and washed 2 times;

(2) 150 μl of blocking solution was added to each well and blocked at 37deg.C for 2.5h;

(3) Taking the serum separated after immunization as an initial dilution multiple, carrying out gradient dilution on 7 concentration points by using a sample diluent by 2 times, adding 100 μl of serum sample into each hole, incubating at 37 ℃ for 90min, and washing for 3 times after incubation is finished;

(4) 100 μl horseradish peroxidase-labeled goat anti-mouse IgG antibody (Jackson, cat# 115-035-003, diluted 30000 times with enzyme-labeled diluent) was added to each well, incubated at 37deg.C for 45min, and washed 5 times after incubation;

(5) Adding 100 μl of color development liquid into each hole, incubating at 37deg.C in dark for 15min, adding stop solution, and measuring OD value at 450nm wavelength;

(6) And judging that the ratio of the OD value of the sample to the OD mean value of the blank control is greater than or equal to 2.1 according to the method development parameters, namely judging that the antigen-specific IgG antibody titer of the serum sample is higher than or equal to the maximum serum dilution corresponding to 2.1 (the OD mean value of the negative control is smaller than 0.05 and is calculated by 0.05).

16.4 cytokine detection

The method for detecting the intracellular cytokines is specifically as follows (wherein the centrifugal operation conditions are 4 ℃ C., and the centrifugation is carried out for 5min at 350 g):

(1) The mice were sacrificed at 28 days after the last immunization (D49) after cervical spine removal, immediately immersed in medical alcohol, transferred to a biosafety cabinet to take out spleen, lysed erythrocytes to prepare spleen cell suspension, counted and adjusted to 1X 10 concentration of spleen cells ⁶ Individual/ml;

(2) To the sterile flow tube was added 1ml of the corresponding group of spleen cell suspensions (i.e., 1X 10 per tube ⁶ Individual cells), 20 μg/ml of stimulatory protein and CD28 (Biolegend, cat: 102102 CD49d (Biolegend, cat: 103708 Brefeldin a (Biolegend, cat: 420601 At 37deg.C with 5% CO ₂ Incubating for 18h in a constant temperature incubator;

(3) After the incubation, the flow tube was removed, washed twice with DPBS (Lonza, cat# 17-512F), and dye Fixable Viability Stain 450 (BD Biosciences, cat# 562247) (1:100) was added and incubated at 4℃for 20min in the absence of light;

(4) Wash buffer (WB, 1% FBS-PBS buffer) was prepared: adding 5ml of inactivated FBS into 495ml of PBS, shaking and mixing uniformly, and preparing at present;

(5) Washing with 1ml Wash buffer for 2 times, adding TruStainFcX ^TM Blocking agent (Biolegend, cat# 103710) (1:40), incubation at 4℃for 10min; CD3e-FITC (BD Biosciences, cat# 553062) (1:100), CD4-APC-Cy7 (BD Biosciences, cat# 552051) (1:100), CD8a-PE-Cy7 (BD Biosciences, cat# 552877) (1:100) were then added for surface staining, and incubated at 4℃for 20min;

(6) Washing with 1ml Wash Buffer for 2 times, adding Fixation Buffer (Biolegend, cat# 420801), fixing, and incubating at room temperature in dark place for 20min;

(7) Film breakers (PWB, 1×) were formulated: operating in biosafety cabinet, sucking 10ml Intracellular Staining Permeabilization Wash Buffer (10×) (Biolegend, cat# 421002) into 90ml deionized water, mixing thoroughly;

(8) Washing with 1ml Wash buffer for 2 times, adding 1ml PWB (1×), centrifuging, performing cell membrane disruption, adding 80 μl PWB (1×) to resuspend cells, gently mixing, and incubating at room temperature in the dark for 5min;

(9) Cytokine staining was performed by adding IL-2-BV605 (BD Biosciences, cat# 563911) (1:100) and IFN-gamma-APC (BD Biosciences, cat# 554413) (1:100) fluorescent antibodies, respectively, and incubating at 4℃for 1h;

(10) Cells were resuspended by adding 100. Mu.l DPBS per tube and collected by flow cytometry after washing 2 times with 1ml Wash buffer.

16.5IgA detection

(1) Taking blood from the mice 14 days after the last immunization (D35), dissolving the freeze-dried standard substance according to the instruction of Mouse IgA ELISA Kit (Sizhengbai organism, cat# MCD-000-090), standing for 15 minutes, and uniformly mixing for later use;

(2) 2 times of the standard working solution is subjected to gradient dilution for 7 concentration points, and a serum sample to be tested is diluted by a proper time by a sample diluent according to a pre-experiment result;

(3) Adding standard substances and samples at 100 mu L/hole according to an addition plate graph, sealing a plate by using a sealing plate membrane, and incubating for 2 hours at room temperature in an oscillating way;

(4) Preparing a 1 XWash buffer: 20ml of 20 XWash buffer is sucked and added into 380ml of water for injection, and the mixture is used after shaking and mixing uniformly, and is prepared at present;

(5) After the incubation is finished, the ELISA plate is taken off from the oscillator, the solution in the hole is discarded, and 350 mu l of 1 XWash buffer is added into each hole for washing 5 times, and each time is soaked for 30s;

(6) And diluting the enzyme-labeled antibody by using an enzyme-labeled antibody diluent at a ratio of 1:100. Mu.l of diluted enzyme-labeled antibody was added to each well and incubated for 1 hour at room temperature with shaking. The solution in the wells was discarded, washed 5 times with 350. Mu.l of 1 XWash buffer, and soaked for 30s each time;

(7) Adding 100 mu L of color-developing agent into each hole, sealing by a sealing plate membrane, and incubating for 20-25min at room temperature in dark place;

(8) 100. Mu.L of stop solution was added to each well, and OD450 was measured immediately after mixing.

16.6 test results

(1) General clinical observations

During the test period, no abnormal state or death of the tested animals occurs, which indicates that the vaccine has good safety.

(2) T cell immune effect investigation

As shown in FIGS. 7-8, each group was induced to produce a specific cytokine IL-2 after immunization ⁺ 、IL-2 ⁺ IFN-γ ⁺ And IFN-gamma ⁺ Wherein Gr1 immunization produces specific IL-2 ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequencies were 0.23%, 0.1%, 0.049%, respectively, and Gr2 immunization produced specific IL-2 ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequencies of 0.06%, 0%, 0.051% respectively, gr3 immunization produced specific IL-2 ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequencies were 0.38%, 0.43%, 0.088%, respectively, and Gr4 immunization produced specific IL-2 ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequency was 0.21%, 0.079% and 0.05%, respectively. From this, it can be seen that each cytokine generated by Gr3 immunization is higher than Gr1 and Gr2, and there is a statistical difference, indicating that the immune effect of AS01 adjuvant is better than the other two adjuvants; gr1 and Gr4 used the same adjuvant, and there was no statistical difference in cytokines.

(3) Investigation of IgG antibody Induction Effect

The test results are shown in FIGS. 9-10, and specific antibody IgG can be induced after immunization of each group. Wherein, when the coated gB1-P antigen is detected, the geometric average titer (GMT) of the IgG antibody induced after Gr1 immunization of the mice is 1600000, the GMT of the IgG antibody induced by Gr2 immunization is 46166, the GMT of the IgG antibody induced by Gr3 immunization is 11596475, the GMT of the IgG antibody induced by Gr4 immunization is 334605, and the titer of the IgG antibody induced by Gr3 is higher than that of Gr1, gr2 and Gr4, and the statistical difference exists. When the coated gB1-fusion antigen is detected, the IgG antibody GMT induced after Gr1 immunization of mice is 242515, the IgG antibody GMT induced by Gr2 immunization is 757.9, the IgG antibody GMT induced by Gr3 immunization is 36755835, and the IgG antibody GMT induced by Gr4 immunization is 278576, so that the titer of the IgG antibody induced by Gr3 is still higher than that of Gr1, gr2 and Gr4, and the statistical difference exists, but no significant difference exists between Gr1 and Gr 4.

(4) Investigation of IgA antibody Induction Effect

As shown in FIG. 11, specific IgA antibodies were induced in each group after immunization, wherein the Geometric Mean Concentration (GMC) of IgA antibodies induced after Gr1 immunization was 40.75, the IgA antibodies induced by Gr2 immunization was 64.65, the IgA antibodies induced by Gr3 immunization was 41.66, and the IgA antibodies induced by Gr4 immunization was 90.58. It can be seen that the IgA antibody concentration produced by Gr4 immunization is higher than that produced by Gr2 immunization, and that Gr2 produces IgA at a higher concentration than that of Gr1 and Gr3, and that there are statistical differences from each other.

16.7 conclusion

(1) According to the detection results of the cytokines and the IgG antibodies, each group shows better immunogenicity in the test, which indicates that the vaccine has good immunity; wherein Gr3 (gB 1-P+AS 01) produces an immune response stronger than Gr1 (gB 1-P+CpG+Al (OH) ₃ ) And Gr4 (gB 1-fusion+CpG+Al (OH) ₃ ) And is stronger than Gr2 (gB 1-P+CpG), indicating that the induction effect of AS01 adjuvant on cell immunity and IgG antibody is better than CpG or CpG and Al (OH) ₃ Is a combination of (a) and (b).

(2) According to IgA antibody detection results, each group shows good immunogenicity in experiments, which shows that the vaccine has good immunity; wherein Gr4 (gB 1-fusion+CpG+Al (OH) ₃ ) The resulting immune response is stronger than Gr2 (gB 1-P+CpG) and than Gr1 (gB 1-P+CpG+Al (OH) ₃ ) And Gr3 (gB 1-P+AS 01), indicating that gB1-fusion antigen+CpG+Al(OH) ₃ The induction effect on IgA antibodies was strongest, but specific adjuvant combinations did not show outstanding immune effects.

Example 17 in vitro neutralization assay of HSV vaccine comprising different adjuvants

20C 57BL/6N mice (females, 6-8 weeks old, purchased from Beijing Vitrehua laboratory animal technologies Co., ltd.) were randomly divided into 4 groups of 5 mice each, and group immunization was performed in the manner shown in Table 7. Taking blood 14 days after secondary immunization, wherein the blood taking method is that blood is taken from the ocular venous plexus, and the blood taking amount is 300-500 mu l each time; after blood collection, the sample was not anticoagulated, placed at 37℃for 1 hour, then at 4℃for 0.5 hour, centrifuged at 8000rpm for 10 minutes, serum was collected, inactivated in a water bath at 56℃for 30 minutes, stored at-20℃and subjected to neutralization tests for different strains, respectively, in the manner shown in Table 7.

TABLE 7 grouping experiment design cases

Group of	Vaccine working fluid	Immunization dose	Immunization pathway and volume	Immunization program	Challenge strains
						1	Gr1	1/10HD	Intradermal 0.1ml	2 doses, interval 21d	HSV-1syn
2	Gr3	1/10HD	Intradermal 0.1ml	2 doses, interval 21d	HSV-1syn
						3	Gr4	1/10HD	Intradermal 0.1ml	2 doses, interval 21d	HSV-1syn
4	Gr3	1/10HD	Intradermal 0.1ml	2 doses, interval 21d	HSV-2G

17.1 serum neutralization Activity assay

In vitro neutralization assays were performed with the serum collected as described above and with the corresponding strains, and the results are shown in FIG. 12. In the neutralization test of the HSV-1syn strain, the neutralization capacity after being immunized by Gr3 is strongest, the Geometric Mean Titer (GMT) reaches 403.6, which is higher than Gr1 and Gr4, the neutralization capacity after being immunized by Gr4 is weakest, and the three groups are in sequence statistically different. In the neutralization test of the HSV-2G strain, the Gr3 with the best immunization effect is adopted for the neutralization test, and the result is shown in figure 13, the Gr3 can induce stronger neutralization capability to the HSV-2G strain after immunization, and the geometric average titer (GMT) can reach 97.14.

17.2 plaque reduction neutralization assay

(1) HSV-1 neutralization assay

Vero cells were cultured in about 4X 10 medium containing 10% fetal bovine serum ⁵ Inoculating the cell culture medium into 24-well plates with the volume of each volume of 500 mu L of each well, culturing overnight in a cell culture box at 37 ℃ to form a cell monolayer, and after the cells are about 90% -100%, discarding the supernatant, and washing with PBS for later use; diluting serum to be tested according to a certain proportion, uniformly mixing 240 mu l of diluted serum with 1200pfu/ml HSV-1 mu l, incubating for 30 minutes at room temperature, and simultaneously setting a positive control sample incubated by mixing a culture medium and a strain; removing culture supernatant from 24-well plate Vero E6 cells, inoculating the above serum+virus mixed system into Vero E6 cells at a concentration of 200 μl/well, 2 duplicate wells each, and CO ₂ A cell incubator for 2 hours; removing incubation liquid in 24-well plate, adding 0.5% low-melting agarose 500 μl/well, cooling 24-well plate in refrigerator at 4deg.C for 10min, and adding CO after agarose solidifies ₂ A cell incubator for 72 hours; after plaque is formed, 400 μl/hole of 4% paraformaldehyde is added for fixation, the fixed 24-pore plate is washed once with pure water after the room temperature is over night, agarose is made to fall off, 300 μl of 0.2% crystal violet staining solution is added into each hole, and after the room temperature is stained for 4 hours, the solution is washed three times with pure water; the number of plaques formed per well was recorded and the neutralization titers of the sera were calculated from the ratio of the number of plaques containing serum to the number of plaques of the positive control that did not contain serum.

The results of the experiment are shown in Table 8, and both Gr1 and Gr3 serum samples produced a certain neutralization potency for HSV-1, and Gr3 produced a neutralization potency significantly higher than Gr1.

TABLE 8 neutralization titers of different serum samples on HSV-1 strains

(2) HSV-2 neutralization assay

HSV-2 neutralization test was performed according to the above HSV-1 method using Gr3 having a good neutralizing effect on HSV-1, and the results are shown in Table 9, gr3 also being capable of producing a high neutralizing titer on HSV-2.

TABLE 9 neutralization titers of different serum samples on HSV-2 strains

In summary, various formulas of the vaccine have good immunity performance against both HSV-1 and HSV-2 strains; wherein Gr3 (gB 1-P+AS 01) can generate stronger neutralization capacity against both HSV1 and HSV2 strains, and is significantly better than Gr1 (gB 1-P+CpG+Al (OH) ₃ ) And Gr4 (gB 1-fusion+CpG+Al (OH) ₃ ) Shows that the neutralization capacity of AS01 adjuvant to strains is better than CpG+Al (OH) ₃ Is a combination of (a) and (b).

EXAMPLE 18 HSV vaccine Immunity test comprising different antigens

T cell immune effect

Immunization programs for vaccination were used for days 0 and 21, and C57BL/6N mice were immunized at 5. Mu.g/mouse. Spleen cells were isolated 14 days after the last immunization (D35), stimulated with 20. Mu.g/ml of 5 immune proteins (gB 1-WT, gB1-P, gB1-fusion, gB2-fusion and gB1-gB 2-fusion), respectively, and examined for levels of antigen-specific cytokines induced by recombinant human herpes simplex virus vaccine by intracellular cytokine detection as described in example 16. As shown in FIG. 14, immunization with the gB1-WT antigen resulted in specific IL-2 upon the use of AS01 adjuvant ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequency was 0.21%, 0.079%, respectively, and specific IL-2 was produced by immunizing gB1-P antigen ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequencies were 0.47%, 0.63%, 0.18%, respectively, and specific IL-2 was produced by immunizing gB1-fusion antigen ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequency was 0.36%, 0.13%, respectively, immunizationgB2-fusion antigen produces specific IL-2 ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequencies were 0.46%, 0.65%, 0.22%, respectively, and specific IL-2 was produced by immunizing gB1-gB2-fusion antigen ⁺ 、IL-2 ⁺ IFN-γ ⁺ 、IFN-γ ⁺ Factor cell occupancy of CD4 ⁺ T cell frequency was 0.43%, 0.73% and 0.27%, respectively. The gB1-gB2-fusion antigen combined with AS01 adjuvant was immunized with cytokine higher than other immunized groups, but there was no statistical difference with gB1-P, gB1-fusion and gB2-fusion, and there was a statistical difference with gB 1-WT.

(II) antigen-specific IgG antibody detection

Immunization programs for vaccination were used for days 0 and 21, and C57BL/6N mice were immunized at 5. Mu.g/mouse. Blood was collected 14 days after the last immunization (D35), and antigen-specific IgG antibody titer was measured according to the method of example 16. FIG. 15 shows that, on the basis of AS01 adjuvant, the antigens gB1-WT, gB1-P, gB1-fusion, gB2-fusion and gB1-gB2-fusion induced the body to produce antigen-specific IgG antibody titers GMT of 4525483, 4935075, 3200000, 2934413 and 5868826, respectively. The gB1-gB2-fusion antigen produced IgG antibody titers higher than gB1-P and gB1-WT, but no statistical differences, higher than gB1-fusion and gB2-fusion, and statistical differences.

EXAMPLE 19 in vitro HSV neutralization assay of different antigens

Immunization programs for vaccination were used for days 0 and 21, and C57BL/6N mice were immunized at 5. Mu.g/mouse. Blood was collected 14 days after the last immunization (D35), and antigen-specific neutralizing antibody titer was detected by the method of example 17. FIG. 16 shows that, on the basis of AS01 adjuvant, gB1-WT, gB1-P, gB1-fusion and gB1-gB2-fusion antigens induced the body to produce HSV-1 strain antigen-specific neutralizing antibody titres GMT of 787.2, 575.1, 554.6 and 832.5, respectively, with no statistical difference between the antigens, but the gB1-gB2-fusion antigen-induced neutralizing antibody titres GMT was highest, suggesting that fusion of different serotypes of HSV protein may enhance immune effects and thus cross protection against different strains.

The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims

1. An HSV immunogenic recombinant protein, wherein the recombinant protein comprises, or is at least 90% identical in sequence to, a gB protein ectodomain of at least one of HSV-1 and HSV-2, or a functional fragment thereof; wherein the functional fragment comprises at least domains I and II of the extracellular domain of the gB protein of at least one of HSV-1 and HSV-2.

2. The HSV immunogenic recombinant protein of claim 1, wherein the recombinant protein comprises an extracellular domain of a gB protein of HSV-1 or a functional fragment thereof and an extracellular domain of a gB protein of HSV-2 or a functional fragment thereof.

3. The HSV immunogenic recombinant protein of claim 2, wherein the C-terminus of the gB protein ectodomain of HSV-1 or a functional fragment thereof is directly or indirectly linked to the N-terminus of the gB protein ectodomain of HSV-2 or a functional fragment thereof, or the C-terminus of the gB protein ectodomain of HSV-2 or a functional fragment thereof is directly or indirectly linked to the N-terminus of the gB protein ectodomain of HSV-1 or a functional fragment thereof.

4. The HSV immunogenic recombinant protein of claim 1, wherein the gB protein ectodomain or functional fragment thereof is mutated for at least one amino acid.

5. The HSV immunogenic recombinant protein of claim 1, wherein the amino acid sequence of the recombinant protein is at least 90% identical to any one of SEQ ID nos. 1-7.

6. The method for preparing an HSV immunogenic recombinant protein according to any one of claims 1 to 5, comprising the steps of:

s3: purifying the culture product to obtain the recombinant protein.

7. Use of the HSV immunogenic recombinant protein of any one of claims 1-5 in the preparation of a vaccine for preventing HSV infection.

8. Use according to claim 7, wherein the recombinant protein is used in a single vaccine dose of 20-80 μg, preferably 50 μg.

9. The use according to claim 8 wherein the recombinant protein is used in combination with at least one immunoadjuvant comprising at least one of aluminium adjuvants, squalene, tocopherol, MPL, LPA, cpG, poly (I: C) and QS-21.

10. The use according to claim 9, wherein the human dose of recombinant protein is 40-160 μg, dispersed in two doses of the same dose vaccine administered 14-28 days apart.

11. A vaccine for the prevention of HSV infection, comprising an HSV immunogenic recombinant protein according to any one of claims 1 to 5; the vaccine provides protection against at least one of the two serotypes HSV-1 and HSV-2.

12. Vaccine according to claim 11, characterized in that the single dose of vaccine contains 20-80 μg, preferably 50 μg of said recombinant protein.

13. The vaccine of claim 11, further comprising an immunoadjuvant, the immunoadjuvant comprising at least one of aluminum adjuvants, squalene, tocopherol, MPL, LPA, cpG, poly (I: C), and QS-21.

14. Vaccine according to claim 13, wherein the immunoadjuvant comprises MPL and QS-21, preferably a single dose of vaccine comprises 10-100 μg MPL and 10-100 μg QS-21.

15. The vaccine of any one of claims 11-14, wherein the vaccine comprises a first dose and a second dose of the same composition administered at intervals of 14-28 days.