Tri-antigen I-type herpes simplex virus subunit vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of vaccine preparation, and discloses a three-antigen I-type herpes simplex virus subunit vaccine as well as a preparation method and application thereof, and the vaccine comprises herpes simplex virus recombinant protein and a composite adjuvant; the herpes simplex virus recombinant protein comprises extracellular domains of three proteins, namely HSV (herpes simplex virus)-1gC, gD and gE, wherein the amino acid sequences of the extracellular domains of the HSV-1gC, gD and gE are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and the composite adjuvant is composed of CpG oligodeoxynucleotide and QS-21. Comparison experiments prove that humoral immune response and cellular immune response levels induced by the subunit vaccine combined by the CpG 1018S + QS-21 composite adjuvant are equivalent to immune response levels induced by mRNA vaccines, and the neutralizing effect of serum of mice inoculated with the subunit vaccine is also equivalent to that of serum of mRNA group mice. And a foundation is laid for popularization of subunit vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine preparation, and particularly relates to a type I herpes simplex virus subunit vaccine with three antigens, and a preparation method and application thereof. Background Art

[0002] The most common symptom of herpes simplex virus type 1 (HSV-1) infection is inflammation of the lips, called cold sores; when the infection is severe, it may cause conjunctivitis, which is the main cause of infectious blindness in developed countries; HSV-1 infection is also associated with the development of Alzheimer's disease; and HSV-1 is a neurotropic virus with the ability to invade nerve cells and cause damage to the nervous system, resulting in herpes simplex virus encephalitis. When infecting the nervous system, HSV-1 usually survives in the host in a latent infection form until the virus is reactivated. This form of infection enables the virus to evade the defense of the host immune system. The human trigeminal ganglion is the main latent site of HSV-1, and once the virus infection is established, it is very difficult to eliminate. Currently, there is no vaccine against herpes simplex virus approved for marketing, but there are a variety of vaccines in clinical trials.

[0003] Herpes simplex virus has a complex entry mechanism. Among the viral membrane proteins on its outermost lipid bilayer envelope, gB, gC, gD, gE, gH, and gL are crucial for the virus to enter host cells and for viral immune escape. gD is an important receptor-binding protein that can recognize and bind to specific receptors on the surface of host cells, and this interaction is crucial for virus fusion and subsequent infection. gD is the most abundant glycoprotein present on the viral membrane surface and contains multiple neutralizing epitopes, which makes gD the most common target for vaccine development. The gC protein can bind to C3b complement to inhibit complement activation and participate in immune escape. The glycoproteins gE and gI exist in the form of a heterodimer and play an important role in the viral life cycle. gE can bind to the Fc domain of IgG, interfere with the binding of C1q to the antigen-antibody complex, thereby blocking complement activation and antibody-dependent cell-mediated cytotoxic activity. In the HSV-1 vaccine formulation, using gD, gC, and gE as immunogens may increase the protective effect of the vaccine. gC and gD have the effect of neutralizing the virus, gD and gE have the effect of blocking the intercellular spread of the virus, and gC and gE have the effect of preventing immune escape. Summary of the Invention

[0004] In order to overcome the problems of the prior art, the present invention provides a type I herpes simplex virus subunit vaccine with three antigens, and a preparation method and application thereof, aiming to improve the immunogenicity, specificity, and immune functions such as virus clearance of the type I herpes simplex virus subunit vaccine.

[0005] The object of the present invention is achieved as follows:

[0006] The first aspect of the present invention provides a type I herpes simplex virus subunit vaccine of three antigens, wherein the vaccine comprises a herpes simplex virus recombinant protein and a compound adjuvant;

[0007] The herpes simplex virus recombinant protein comprises the extracellular domains of three proteins, namely HSV-1 gC, gD, and gE. The amino acid sequences of the extracellular domains of HSV-1 gC, gD, and gE are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively; the compound adjuvant consists of CpG oligodeoxynucleotide and QS-21.

[0008]

[0009]

[0010]

[0011] Further, the mass ratio of CpG oligodeoxynucleotide to QS-21 in the composite adjuvant is 1:1.

[0012] Further, the concentrations of the three proteins, HSV-1 gC, gD, and gE, in the vaccine are all 5 μg per injection, and the addition amount of the composite adjuvant is 10 μg per injection.

[0013] The second aspect of the present invention provides a preparation method of the type I herpes simplex virus subunit vaccine of the three antigens described in the first aspect, including the following steps:

[0014] 1) Clone the coding gene sequences corresponding to the extracellular domains of the three proteins, HSV-1 gC, gD, and gE, between the XhoI and EcoRI restriction enzyme cleavage sites of the pBlueScript II SK(+) vector to construct a recombinant pBlueScript II SK(+) vector;

[0015] 2) Then, obtain the target fragments of the coding genes of the three proteins from the above recombinant pBlueScript II SK(+) vector and insert them into the pATX2 vector to construct a recombinant pATX2 vector;

[0016] 3) Use mammalian system CHO cells to express the proteins respectively, and purify the proteins by affinity chromatography to obtain the extracellular domains of the three proteins, HSV-1 gC, gD, and gE, respectively;

[0017] 4) Use QS-21 and CpG oligodeoxynucleotide as the composite adjuvant of the subunit vaccine; the mass of each of the proteins HSV-1 gC, gD, and gE in the vaccine is 5 μg, dissolve it in phosphate buffer to make 15 μg per animal per injection, and add 5 μg of CpG oligodeoxynucleotide and 5 μg of QS-21 to the injection before immunization, thereby preparing the type I herpes simplex virus subunit vaccine of the three antigens.

[0018] The third aspect of the present invention provides the application of the type I herpes simplex virus subunit vaccine of the three antigens described in the first aspect, or the preparation method described in the second aspect, and the application includes:

[0019] 1) Prepare a pharmaceutical composition for preventing or treating diseases caused by herpes simplex virus infection;

[0020] 2) Prepare a pharmaceutical composition for preventing or treating herpes simplex virus infection.

[0021] Further, the herpes simplex virus subunit vaccine is immunized by intramuscular, subcutaneous or intradermal injection; preferably, it is an intramuscular injection.

[0022] The fourth aspect of the present invention provides the application of a complex adjuvant composed of CpG oligodeoxynucleotide and QS-21 in enhancing the immune function of herpes simplex virus subunit vaccine.

[0023] The advantages and beneficial effects of the present invention are as follows:

[0024] 1. In the type I herpes simplex virus subunit vaccine with three antigens of the present invention, CpG oligodeoxynucleotide and QS-21 are used as a complex adjuvant, significantly improving the levels of humoral immune response and cellular immune response induced by the subunit vaccine;

[0025] 2. mRNA vaccines have relatively strict requirements for conditions during production quality control and cold chain transportation. Therefore, subunit vaccines have certain advantages, but the immune response level of traditional subunit vaccines is poor. Through comparative experiments, it is confirmed in this application that the levels of humoral immune response and cellular immune response induced by the subunit vaccine using the CpG 1018S+QS-21 complex adjuvant combination are equivalent to those induced by mRNA vaccines, and the neutralization effect of the sera of mice after inoculating the subunit vaccine is also equivalent to that of the sera of mice in the mRNA group, laying a foundation for the popularization of subunit vaccines;

[0026] 3. The type I herpes simplex virus subunit vaccine with three antigens of the present invention can not only induce an effective specific IgG antibody titer against gE, but also effectively clear the viral load in the heart, liver, spleen, lungs, kidneys, spinal cord, brain, and trigeminal nerve tissues, and has a therapeutic effect equivalent to that of the type I herpes simplex virus mRNA vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the drawings and embodiments.

[0028] Figure 1 Showing the detection results of the LNP-mRNA vaccine described in Comparative Example 1; wherein, A: particle size; B: dispersion coefficient; C: encapsulation efficiency;

[0029] Figure 2 Showing the IgG responses of each antigen and the detection results of neutralizing antibodies in mice in Test Example 1; wherein, A: gD-specific IgG response; B: gE-specific IgG response; C: gC-specific IgG response; D: geometric mean titer detection results;

[0030] Figure 3 Showing the ELISA detection results in Test Example 1; wherein, A: IL-2 concentration in the supernatant of spleen cells; B: IFN-γ concentration in the supernatant of spleen cells;

[0031] Figure 4Show the ELISPOT detection results in Test Example 1; wherein, A: the number of spots of IL-2; B: the number of spots of IFN-γ; C: a representative photo of the ELISPOT reaction of IL-2; D: a representative photo of the ELISPOT reaction of IFN-γ.

[0032] Figure 5 Show the flow cytometry detection results in Test Example 1; wherein, A: the proportion of CD4+ T cells producing IL-2 in mouse spleen cells; B: the proportion of CD4+ T cells producing IFN-γ in mouse spleen cells; C: a representative image of the flow cytometry detection results.

[0033] Figure 6 Show the q-PCR detection results in Test Example 2;

[0034] Figure 7 Show the HE staining results in Test Example 2. Detailed implementation manners

[0035] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners according to the above-mentioned invention content still fall within the protection scope of the present invention.

[0036] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0037] The present invention will be described in detail below through examples. It should be understood that the following examples are only used for further detailed explanation and illustration of the content of the present invention by way of example, and are not used to limit the present invention.

[0038] In the following examples, CpG 1018S was purchased from Sangon Biotech (Shanghai), and QS-21 was purchased from AlphaDiagnoesticInternational.

[0039] Example 1: Preparation of a type I herpes simplex virus subunit vaccine with three antigens

[0040] This embodiment provides a type I herpes simplex virus subunit vaccine with three antigens, containing a herpes simplex virus recombinant protein and a complex adjuvant; the herpes simplex virus recombinant protein includes the extracellular domains of three proteins, HSV-1 gC, gD, and gE, and the amino acid sequences of the extracellular domains of HSV-1 gC, gD, and gE are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively; the complex adjuvant consists of CpG 1018S and QS-21.

[0041] The preparation method of the type I herpes simplex virus subunit vaccine includes the following steps:

[0042] 1) Clone the coding gene sequences corresponding to the extracellular domains of the three proteins, HSV-1 gC, gD, and gE, between the XhoI and EcoRI restriction enzyme sites of the pBlueScript IISK(+) vector to construct a recombinant pBlueScript II SK(+) vector.

[0043] 2) Then, obtain the target fragments of the coding genes of the three proteins from the above recombinant pBlueScript II SK(+) vector and insert them into the pATX2 vector to construct a recombinant pATX2 vector; a 6*His tag is added to the C-terminus of the target fragment.

[0044] 3) Express the proteins using mammalian system CHO cells and purify the proteins with nickel columns to obtain the extracellular domains of the three proteins, HSV-1 gC, gD, and gE respectively.

[0045] 4) Use QS-21 and CpG 1018S as the complex adjuvant of the subunit vaccine; the mass of each of the proteins HSV-1 gC, gD, and gE in the vaccine is 5 μg, dissolved in phosphate buffer, formulated into 15 μg / animal / dose, and 5 μg of CpG 1018S and 5 μg of QS-21 are added to the dose before immunization, thereby preparing the type I herpes simplex virus subunit vaccine with three antigens.

[0046] Comparative Example 1: Preparation of a type I herpes simplex virus mRNA vaccine with three antigens

[0047] This comparative example provides a type I herpes simplex virus mRNA vaccine with three antigens, which includes mRNA sequences encoding the extracellular domains of the three proteins, HSV-1 gC, gD, and gE. The mRNA is encapsulated with LNP to prepare an LNP-mRNA vaccine for use as a comparative example.

[0048] The preparation method of the type I herpes simplex virus mRNA vaccine with three antigens is as follows:

[0049] In vitro, the synthesized DNA sequence was transcribed into mRNA. After purification by magnetic beads, the mRNA concentration was measured, and the self-made lipid nanoparticles (LNP) were used to encapsulate the mRNA vaccine (volume ratio 1:3).

[0050] The mass of mRNA encoding each extracellular vesicle of the protein (HSV-1 gC, gD, gE) was 5 μg, which was dissolved in phosphate buffer (PBS, pH 7.4) to prepare a type I herpes simplex virus mRNA vaccine (LNP-mRNA vaccine) with three antigens at 15 μg / animal / dose.

[0051] The particle size, polydispersity index (PDI), and encapsulation efficiency of the nanoparticles formed by the LNP-mRNA vaccine were detected, and the detection results are as Figure 1 shown.

[0052] Test Example 1:

[0053] To compare the immune levels of the subunit vaccine and mRNA vaccine described in Comparative Example 1 and Comparative Example 1, the following animal experiments were carried out in this test example. At the same time, the attenuated live HSV-1 vaccine group was used as a control (for the preparation method, see Xingli Xu et al., A HSV 1 mutant leads to an attenuated phenotype and induces immunity with aprotectiveeffect; https: / / pubmed.ncbi.nlm.nih.gov / 32776994 / ).

[0054] Female BALB / c mice at 6-8 weeks of age free of HSV-1 pathogens provided by the Experimental Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences were randomly divided into 4 groups, with 6 mice in each group. They were raised under SPF conditions and allowed to eat and drink freely.

[0055] The subunit vaccine and mRNA vaccine in Example 1 and Comparative Example 1 were taken respectively and used to immunize mice by intramuscular injection, 50 μL / mouse, and the blank group was injected with an equal amount of PBS. The virus titer of the attenuated live HSV-1 vaccine group was 10,000 PFU / mouse, and the mice were administered by intranasal drip, 20 μL / mouse. Every 4 weeks, the injection was carried out twice. Two weeks after the last immunization, the mice were anesthetized by intraperitoneal injection of tribromoethanol, and then blood was collected by cardiac puncture and the spleen was collected. The blood was placed at 4 °C overnight and then centrifuged at 3000 rpm for 20 minutes to obtain serum. A single-cell suspension of splenocytes was obtained by grinding the spleen.

[0056] The antibody titers specific for gC, gD, and gE were detected by indirect enzyme-linked immunosorbent assay. The results are as Figure 2As shown in A, B, and C, after two immunizations, the subunit vaccine described in Example 1 using CpG 1018S and QS-21 complex adjuvant could induce an antibody titer of specific gD comparable to that of the mRNA vaccine described in Comparative Example 1 (p>0.05), and the subunit vaccine described in Example 1 could induce higher antibody titers specific for gC and gE than the mRNA vaccine described in Comparative Example 1 (p≤0.001). The attenuated live HSV-1 vaccine administered by nasal drops failed to elicit an effective humoral immune response.

[0057] Mouse sera were inactivated at 56°C for 30 minutes and then serially diluted at a ratio of 1:4:8:16:32:64:128. A negative control group without virus and serum and a positive control group containing only virus were set up. The HSV-1 Mckrae virus strain was diluted with DMEM medium, and the serum and virus were incubated at a ratio of 1:1 for 1 hour at 37°C. After incubation, the virus and serum mixture was added to Vero cells, and the virus solution was removed after 1 hour of incubation. After adding serum-free DMEM medium to wash the cell surface, Vero cells were cultured in DMEM containing 2% newborn bovine serum at 37°C for 4-7 days, and the cytopathic effect was observed daily. Consistent with the antibody titer detection results, the GMTs of the mRNA vaccine group described in Comparative Example 1 and the subunit vaccine group described in Example 1 were similar, being 11.31 and 14.25 respectively, and the GMT of the attenuated live HSV-1 vaccine group was 4, which was lower than the other two groups.

[0058] The double antibody sandwich ELISA method was used to detect the amounts of extracellularly secreted cytokines IL-2 and IFN-γ. A single spleen cell suspension was obtained by grinding the mouse spleen, and the spleen cell concentration was adjusted to 1×10 7 cells / mL by adding RPMI 1640 medium. 100 μL of spleen cells (1×10 6 cells per well) were added to a 96-well cell culture plate, and 50 μL of a gC, gD, and gE protein mixture (at a concentration of 15 μg / mL) was added, and the cells were cultured at 37°C in 5% CO 2 for approximately 24 hours. Positive and negative control groups were set up, and the positive control group was added with 10 μL of PMA+. After incubation, the cell culture supernatant was collected, and the cytokine levels were detected by the standard ELISA method.

[0059] The number of IL-2 and IFN-γ cytokines was detected using an ELISPOT-specific culture plate. The culture plate was activated with 75% ethanol and then washed twice with PBS to remove residual ethanol. Specific anti-IL-2 and anti-IFN-γ primary antibodies at a concentration of 2 μg / mL were added, 50 μL per well, and incubated overnight at 4°C. The next day, the antibodies were aspirated, and after washing with complete 1640 medium, fresh complete 1640 medium was added and incubated for 2 hours to block unbound sites. After removing the 1640 medium, 30 μL of cells (3×10 5 spleen cells per well) were added to each well, supplemented with 75 μL of serum-free medium specific for ELISOPT and 50 μL of a mixture of gC, gD, and gE proteins (30 μg / mL). Negative and positive control groups were set up. In the positive control group, 10 μL of PMA+ was added to each well. The culture plate was incubated overnight at 37°C and 5% CO 2 2. On the third day, after centrifugation at 800 g for 5 minutes, the cells were washed with pre-cooled distilled water and PBS, and then anti-IL-2 and anti-IFN-γ secondary antibodies (1 μg / mL), 50 μL per well, were added and incubated for 2 hours at room temperature. After washing, HRP-Streptavidin (1:1500) was added and incubated for 1 hour at room temperature. After washing with PBS, an ELISPOT detection kit was used to visualize the spots, and the reaction was stopped by rinsing with running water.

[0060] The results of ELISA and ELISPOT are shown in Figure 3 、 Figure 4 . The subunit vaccine of Example 1 using CpG 1018S and QS-21 as a combined adjuvant could induce IL-2 and IFN-γ at levels comparable to those of the mRNA vaccine described in Comparative Example 1.

[0061] Flow cytometry was used to detect the number of CD4+ T cells that could secrete IL-2 and IFN-γ. The treated spleen cells (1×10 6 cells per well) and the mixture of gC, gD, and gE proteins (21 μg / mL) were added to a 16-well plate. After culturing at 37°C for 2 hours, Brefeldin A was added to block cytokine secretion and incubated overnight. The next day, the cell suspension was transferred to a 1.5 mL centrifuge tube, and 100 μL of Zombie NIR containing DMSO was added TMThe dye (diluted 1:2000) was incubated for 15 minutes at room temperature in the dark. After washing twice with staining buffer, 50 μL of staining buffer containing 5 μg / mL of CD16 / CD32 antibody was added and incubated for 10 minutes at 4°C. Then, surface staining antibodies diluted with 50 μL of staining buffer, the PerCP / Cyanine 5.5-labeled anti-mouse CD4 antibody, was added and incubated for 30 minutes at 4°C. After washing with staining buffer, the cells were fixed with 4% formaldehyde and incubated for 20 minutes at room temperature in the dark to fix the cell membrane. After washing twice with permeabilization wash buffer and incubating for 5 minutes, 100 μL of intracellular antibodies diluted with permeabilization wash buffer, including PE-conjugated anti-mouse IFN-γ and APC-conjugated anti-mouse IL-2 antibodies, were added for intracellular staining. The cells were incubated for 40 - 60 minutes at room temperature in the dark. Finally, after adding 400 μL of permeabilization wash buffer for washing, 400 μL was added to resuspend the cells.

[0062] Figure 5 The results showed that the proportions of CD4+ T cells capable of producing IL-2 and IFN-γ in the splenocytes of mice inoculated with the subunit vaccine described in Example 1 were both 0.062%, while the proportions of CD4+ T cells capable of producing IL-2 and IFN-γ in the mRNA vaccine described in Comparative Example 1 were 0.0815% and 0.0788% respectively. There was no significant difference between the subunit vaccine described in Example 1 and the mRNA vaccine described in Comparative Example 1 in terms of the ability to induce CD4+ T cells to produce IL-2 and IFN-γ.

[0063] Test Example 2:

[0064] To evaluate the protective effects of the subunit vaccine described in Example 1 and the mRNA vaccine described in Comparative Example 1 against the HSV-1 McKrae virus strain, 6-week-old BALB / c mice were raised in the Biosafety Level 2 Laboratory of the Small Animal Experiment Department of the Institute of Medical Biology, Chinese Academy of Medical Sciences. 28 days after inoculating the two vaccines, the mice were inoculated with the HSV-1 McKrae virus strain at a dose of 1×10 5 PFU by intranasal instillation. The activity status and clinical symptoms of the mice were observed continuously for 6 days, and finally, tissue samples of the mice were collected and stored at -80°C. The observation results showed that on the sixth day after virus challenge, two mice in the positive control group died, while the mice in the other groups survived. The mice in the mRNA group and the subunit vaccine group were similar to those in the blank control group and did not show obvious clinical symptoms. However, four surviving mice in the positive control group showed clinical symptoms such as arched back, hair loss, keratitis, and decreased activity ability.

[0065] The results of q-PCR are as Figure 6As shown, after the attack of HSV-1 Mckrae virus strain, the heart, liver, lung, kidney, brain, spinal cord and trigeminal nerve tissues were removed from the mice. Compared with the positive control group, the viral loads in the tissues of the mice inoculated with the subunit vaccine described in Example 1 and the mRNA vaccine described in Comparative Example 1 were much lower than those in the positive control group. In the heart, liver, spleen, lung and kidney tissues, the viral load in the positive control group was about 5 times that of the subunit vaccine group. In the spinal cord and trigeminal nerve tissues, the viral loads in the positive control group were about 190 times and 500 times that of the subunit vaccine group, respectively. There were significant differences in the viral loads in the heart, liver, spleen, lung and kidney tissues between the mRNA group and the positive control group (p≤0.05). The viral load in the positive control group was about 8 times that of the mRNA vaccine group, and the viral load in the trigeminal nerve tissue of the positive control group was about 200 times higher than that in the mRNA group. Therefore, both the subunit vaccine and the mRNA vaccine have the effect of inhibiting the proliferation of wild-type virus in different tissues and organs of mice, especially in the nervous system, which indicates that both vaccines have good protective effects.

[0066] The results after HE staining are as Figure 7 shown. In the cortex and hippocampal CA2 region of the mice in the positive control group, neuronal atrophy was visible, the cell staining intensity was increased, the nuclear-cytoplasmic boundary was unclear, and the number of shrunken neurons in the cortex was increased (black arrow). Mild local hemorrhage was observed in the brain (green arrow), and local hemorrhage was also observed in the spinal cord tissue (black arrow). The mice in the mRNA group and the subunit group also showed mild pathological changes in their brain tissues. In the mRNA group, partial neuronal shrinkage, increased cell staining and blurred nuclear-cytoplasmic boundary were found in the cortex of the brain tissue (black arrow). In the subunit group, as shown by the black arrow, a large number of neuronal shrinkages were observed in different regions of the cortex and hippocampus, accompanied by increased cell staining and blurred nuclear-cytoplasmic boundary. The spinal cords of the mRNA and subunit groups were similar to those of the blank control group, and no obvious pathological changes were observed. The above pathological findings further indicate that after immunizing mice with HSV-1 mRNA and subunit vaccines, their nerve tissues, especially the brain and spinal cord tissues, can be protected from the infection and damage of wild-type virus.

[0067] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred arrangement, those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A triple-antigen herpes simplex virus type I subunit vaccine, characterized in that: The vaccine comprises a herpes simplex virus recombinant protein and a composite adjuvant; The herpes simplex virus recombinant protein includes the extracellular domains of three proteins: HSV-1gC, gD, and gE. The amino acid sequences of the extracellular domains of HSV-1gC, gD, and gE are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; The composite adjuvant consists of CpG oligodeoxynucleotide and QS-21.

2. The triple-antigen herpes simplex virus type I subunit vaccine according to claim 1, characterized in that: The mass ratio of CpG oligodeoxynucleotide to QS-21 in the composite adjuvant is 1:

1.

3. The triple-antigen herpes simplex virus type I subunit vaccine according to claim 1 or 2, characterized in that: The concentrations of the three proteins HSV-1gC, gD, and gE in the vaccine are all 5 μg / injection, and the added amount of the composite adjuvant is 10 μg / injection.

4. A method for preparing a triple-antigen herpes simplex virus type I subunit vaccine according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: 1) The coding gene sequences corresponding to the extracellular domains of the three proteins of HSV-1 gC, gD, and gE were cloned between the XhoI and EcoRI restriction sites of the pBlueScript II SK(+) vector to construct a recombinant pBlueScript II SK(+) vector; 2) Next, the target fragments of the three protein encoding genes were obtained from the above recombinant pBlueScript II SK(+) vector and inserted into the pATX2 vector to construct a recombinant pATX2 vector; 3) using mammalian CHO cells to express proteins respectively, and purifying the proteins with a nickel column to obtain the three extracellular domains of HSV-1 gC, gD, and gE respectively; 4) QS-21 and CpG oligodeoxynucleotides are used as composite adjuvants for subunit vaccines; each of HSV-1gC, gD, and gE proteins in the vaccine has a mass of 5 μg, which is dissolved in phosphate buffer and prepared into 15 μg / animal / injection. Before immunization, 5 μg of CpG oligodeoxynucleotides and 5 μg of QS-21 are added to the injection to prepare the triple-antigen herpes simplex virus type I subunit vaccine.

5. The triple-antigen herpes simplex virus type I subunit vaccine according to any one of claims 1 to 3, or the use of the preparation method according to claim 4, characterized in that: The applications include: 1) Preparing a pharmaceutical composition for preventing or treating diseases caused by herpes simplex virus infection; 2) Preparing a pharmaceutical composition for preventing or treating herpes simplex virus infection.

6. The use according to claim 5, characterized in that: The herpes simplex virus subunit vaccine is administered by intramuscular, subcutaneous or intradermal injection.

7. Application of composite adjuvant composed of CpG oligodeoxynucleotide and QS-21 in improving the immune function of herpes simplex virus subunit vaccine.

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