Novel three-antigen HSV-2 subunit vaccine as well as preparation method and application thereof

Abstract

The invention relates to a novel three-antigen HSV-2 subunit vaccine as well as a preparation method and application thereof, and belongs to the technical field of biology. The novel three-antigen HSV-2 subunit vaccine comprises antigens and a composite adjuvant, the antigens comprise HSV-2 gB2 envelope glycoprotein, HSV-2 gC2 envelope glycoprotein and HSV-2 gD2 envelope glycoprotein, and the composite adjuvant is CpG oligonucleotide and an aluminum adjuvant; the vaccine provided by the invention can induce a high-level neutralizing antibody, and widens targets for developing a novel multi-target antigen HSV-2 virus vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel triantigen HSV-2 subunit vaccine, its preparation method, and its application. Background Technology

[0002] Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are chronic, highly prevalent viral infections that cause significant morbidity worldwide. Seropositivity rates for both HSV-1 and HSV-2 increase with age. In the 2016 global HSV infection estimates, most HSV-1 infections were acquired in childhood, with seropositivity increasing from 27.4% in infants aged 0–4 years to 67.1% in children aged 10–14 years and 70.7% in adults aged 45–49 years. HSV-2 infections were acquired after first sexual intercourse, with seropositivity increasing from 4.8% in people aged 15–19 years to 20.8% in people aged 45–49 years. HSV-2 is sexually transmitted and is a leading cause of genital ulcer disease (GUD). It also increases the risk of HIV infection, contributing to the HIV epidemic. HSV-1 is typically acquired in childhood through non-sexual contact and causes oral and eye diseases, but it can also cause gastrointestinal dysplasia (GUD) through sexual transmission. Both HSV-1 and HSV-2 can cause neonatal herpes and neurological disorders. Given the prevalence of HSV-1 and HSV-2 infections and the limited existing prevention and control measures, vaccination is the most effective strategy to reduce the global morbidity burden associated with HSV infection. Vaccine strategies include prophylactic vaccination to prevent infection in susceptible populations and therapeutic vaccination for individuals with symptomatic genital HSV-2 infection.

[0003] HSV-1 and HSV-2, both HSV strains, each contain approximately 74 open reading frames and exhibit high overall homology. However, the surface glycoproteins, key candidate vaccine targets, show variation at the DNA level between the two viral strains, ranging from 11.8% to 64.6%. HSV-1 and HSV-2 share a common structure consisting of a large (>84 genes), double-stranded linear DNA genome enclosed in a capsid (nucleocapsid) within a lipid bilayer envelope. The nucleocapsid is tethered to the envelope by an "outer skin," a HSV-specific structure containing virus-encoded proteins; the complete particle is called a viral particle. HSV infection occurs in several discrete steps. Viral particles are produced via HSV glycoproteins, and then the nucleocapsid is transported along microtubules to the nucleus, where viral DNA is released and replicates. Transcription of HSV genes is catalyzed by the host's RNA polymerase II. Early genes encode enzymes involved in DNA replication and envelope glycoprotein biosynthesis, while later genes primarily encode proteins that form viral particles, ultimately leading to the expulsion of mature viral particles from host cells. Productive infection forms vesicular lesions in the mucosal epithelium, after which the virus spreads to sensory neurons, forming latent infection that typically accompanies the host throughout their life. Reactivation of dormant viruses can cause recurrence of disease at or near the primary site of infection.

[0004] Currently, there is no marketed HSV-2 vaccine. The main treatment drugs are acyclovir, valacyclovir, and famciclovir, which can alleviate symptoms but cannot eliminate the virus. Although anti-HSV-2 drugs can inhibit viral replication and maintain the virus in the incubation period, they cannot completely eradicate the virus or prevent frequent reactivation. Therefore, there is an urgent need to develop safe, effective, and novel drugs and combination therapies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel triantigen HSV-2 subunit vaccine, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a novel triantigen HSV-2 subunit vaccine, comprising an antigen and a composite adjuvant, wherein the antigen comprises HSV-2 gB2 enveloped glycoprotein, HSV-2 gC2 enveloped glycoprotein and HSV-2 gD2 enveloped glycoprotein, and the composite adjuvant is a CpG oligonucleotide and an aluminum adjuvant. The amino acid sequence of the HSV-2 gB2 envelope glycoprotein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.4. The amino acid sequence of the HSV-2 gC2 envelope glycoprotein is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.5; The amino acid sequence of the HSV-2 gD2 envelope glycoprotein is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.6.

[0007] Furthermore, the mass ratio of HSV-2 gB2 enveloped glycoprotein, HSV-2 gC2 enveloped glycoprotein, HSV-2 gD2 enveloped glycoprotein, CpG oligonucleotide, and aluminum adjuvant is 5:5:5:50:125.

[0008] A second aspect of this invention provides a method for preparing a novel triantigen HSV-2 subunit vaccine, using the novel triantigen HSV-2 subunit vaccine of claim 1 or 2, characterized by comprising the following steps: The HSV-2 gB2 enveloped glycoprotein, HSV-2 gC2 enveloped glycoprotein, and HSV-2 gD2 enveloped glycoprotein dissolved in protein storage buffer are mixed evenly with aluminum adjuvant and left to stand overnight at 4°C with gentle shaking. Before vaccination, the mixture is then mixed evenly with CpG oligonucleotides to obtain the novel triantigen HSV-2 subunit vaccine.

[0009] Furthermore, the protein storage buffer consists of 50 Mm Tris-HCl, 500 Mm NaCl, 5% glycerol, and pH 8.0.

[0010] Furthermore, when shaking overnight, the speed is 80 rpm / min.

[0011] The third aspect of this invention provides a novel triantigen HSV-2 subunit vaccine prepared by the above-mentioned method.

[0012] The fourth aspect of this invention provides the use of the novel triantigen HSV-2 subunit vaccine in the preparation of drugs for the prevention and / or treatment of HSV-2.

[0013] In this invention, extracellular segments of HSV-2 gB2, HSV-2 gC2, and HSV-2 gD2 are selected, and a GP67 secretion signal peptide sequence is inserted at the N-terminus of each envelope glycoprotein amino acid sequence, and six histidine tags are inserted at the C-terminus of the amino acid sequence.

[0014] In this invention, HSV-2 gB2 envelope glycoprotein, HSV-2 gC2 envelope glycoprotein and HSV-2 gD2 envelope glycoprotein are prepared using a baculovirus-insect cell expression system.

[0015] The dosage of each injection of the novel triantigen HSV-2 subunit vaccine of this invention for immunizing mice is as follows: 5 μg HSV-2 gB2 envelope glycoprotein, 5 μg HSV-2 gC2 envelope glycoprotein, 5 μg HSV-2 gD2 envelope glycoprotein, 50 μg CpG oligonucleotide, and 125 μg aluminum adjuvant.

[0016] In this invention, when preparing the novel triantigen HSV-2 subunit vaccine, the protein antigen solution is thawed on ice, and the required amount is mixed evenly with the required amount of aluminum adjuvant (Alum). The mixture is then placed in a 4°C refrigerator and slowly shaken overnight on a rotating rack to ensure sufficient adhesion between the protein antigen and the aluminum adjuvant. Before vaccination, it is then mixed evenly with CpG oligonucleotides for use in mouse immunization.

[0017] This invention provides a novel triantigen HSV-2 subunit vaccine administered via intramuscular injection, with three doses given two weeks apart. It has application value in the prevention and treatment of HSV-2.

[0018] Compared with the prior art, the beneficial effects of this invention are as follows: This invention employs a different combination of HSV-2 envelope glycoproteins than existing technologies, specifically a combination of HSV-2 gB2, gC2, and gD2. This combination uses the gB2 protein antigen as the core target of the vaccine, and adds the viral immune escape protein gC2 and the viral entry protein gD2 to create a novel triantigen HSV-2 subunit vaccine, aiming to prevent HSV infection through multiple immune pathways. Immunogenicity was validated in mice, and the vaccine induced a high titer of specific IgG antibodies, reaching 1 × 10⁻⁶. 6 The serum from mice immunized with three doses of the vaccine was able to effectively neutralize HSV-1 and HSV-2, providing a new candidate for the development of an HSV-2 vaccine. Attached Figure Description

[0019] Figure 1 The values ​​represent the levels of specific IgG antibodies in mouse serum after three immunizations; where A, B, C, and D represent the levels of specific IgG antibodies against the HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2 gE2 envelope proteins in mouse serum, respectively; Blank represents the blank control well. Figure 2 The values ​​represent the levels of specific IgG subtype antibodies in mouse serum after three immunizations; where A: reflects the level of specific IgG1 antibody in mouse serum; B: reflects the level of specific IgG2a antibody in mouse serum; and C: reflects the difference between the levels of specific IgG1 and IgG2a antibodies in mouse serum. Figure 3The titers of neutralizing antibodies in mouse serum after three immunizations are shown; where A: reflects the titer of neutralizing antibodies against HSV-2 G strain in mouse serum; B: reflects the titer of neutralizing antibodies against HSV-1 17 strain in mouse serum. Figure 4 The image shows the ELISPOT assay for mouse-specific T cell responses after three immunizations. A: Reflects the responsiveness of mouse IFN-γ+ specific T cells; B: Reflects the responsiveness of mouse IL-4+ specific T cells; C: Partial original ELISPOT image. Figure 5 The values ​​represent the cytokine content in the supernatant of mouse spleen cell culture after three immunizations; where A and B reflect the concentrations of Th1 cytokines IFN-γ and IL-2 in the supernatant of mouse spleen cell culture; and C and D reflect the concentrations of Th2 cytokines IL-4 and IL-10 in the supernatant of mouse spleen cell culture. Where: ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the embodiments.

[0021] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0022] 1. Gene sequence origin and composition: The HSV-2 gB2, gC2, and gD2 envelope glycoproteins in the novel triantigen HSV-2 subunit vaccine of this invention are extracted from the extracellular portion. After codon optimization by Kunming Genscript Biotech Co., Ltd., the gene sequences were synthesized and ligated into the pFastBac1 vector through Hind III and EcoRI restriction sites. The resulting HSV-2 gB2pFastBac1 plasmid, HSV-2 gC2 pFastBac1 plasmid, and HSV-2 gD2 pFastBac1 plasmid were double-digested to verify whether the gene fragment insertion position was correct and whether the gene fragment size was consistent with the design size. The pFastBac1 plasmid with the correct inserted gene fragment size and position was sequenced to confirm that the gene inserted into the pFastBac1 plasmid was stable and had not undergone sequence mutation, and the corresponding amino acid sequence was obtained according to the designed gene sequence.

[0023] The amino acid sequence design scheme for the HSV-2 gB2 envelope glycoprotein is: GP67 Signal peptide + gB2 (98-730aa) + His-tag + stop codon. The amino acid sequence of the HSV-2 gB2 envelope glycoprotein is shown in SEQ ID NO.1. The amino acid sequence design scheme for the HSV-2 gC2 envelope glycoprotein is: GP67 Signal peptide + gC2 (28-447aa) + His-tag + stop codon. The amino acid sequence of the HSV-2 gC2 envelope glycoprotein is shown in SEQ ID NO.2. The amino acid sequence design scheme for the HSV-2 gD2 envelope glycoprotein is: GP67 Signal peptide + gD2 (26-310aa) + His-tag + stop codon. The amino acid sequence of the HSV-2 gD2 envelope glycoprotein is shown in SEQ ID NO.3. The nucleotide sequence of the HSV-2 gB2 envelope glycoprotein is shown in SEQ ID NO.4; The nucleotide sequence of the HSV-2 gC2 envelope glycoprotein is shown in SEQ ID NO.5; The nucleotide sequence of the HSV-2 gD2 envelope glycoprotein is shown in SEQ ID NO. 6; 2. Transformation of plasmids containing HSV-2 gB2, gC2, and gD2 gene expression genes respectively: (1) The three pre-designed individual HSV-2 gB2, HSV-2 gC2, and HSV-2 gD2 gene sequences were sent to Kunming GenScript Biotech Co., Ltd. for sequence synthesis. They were then ligated to the pFastBac1 vector plasmid via Hind III and EcoRI restriction sites to obtain HSV-2 gB2pFastBac1, HSV-2 gC2 pFastBac1, and HSV-2 gD2 containing the HSV-2 gB2, gC2, and gD2 gene sequences, respectively. Three different plasmids of pFastBac1 were added to competent DH5α bacteria at a dose of 400 ng each. After mixing, the bacteria were incubated on ice for 30 min, then heat-shocked at 42°C for 45 s. The bacteria were then quickly transferred to ice and incubated on ice for another 2 min. After that, 500 μL of antibiotic-free SOC medium (Solepro: L1020-1L) was added, and the bacteria were shaken at 37°C and 120 rpm for 1 h. 100 μL of the bacterial culture was then spread on ampicillin (Amp) resistant LB (Amp+) solid medium and incubated upside down in an incubator at 37°C for 12 h. Single colonies were picked and inoculated into 10 mL of LB (Amp+) liquid medium and cultured at 37°C and 250 rpm for 8 h. 1 mL of the bacterial culture was sent to a sequencing company for sequencing identification. 10 μL of the remaining bacterial culture was inoculated into 200 mL of LB (Amp+) liquid medium and cultured at 37°C and 250 rpm for 16 h. Plasmids were then extracted using the PureYield plasmid Maxiprep system plasmid extraction kit (Promega: A2393) to obtain three different plasmids: HSV-2 gB2 pFastBac1, HSV-2 gC2 pFastBac1, and HSV-2 gD2 pFastBac1.

[0024] The formulation of 200mL ampicillin-resistant LB (Amp+) solid medium is as follows: 2g tryptone, 1g yeast extract, 2g sodium chloride, 4g agar powder, 20mg ampicillin, and 200mL ultrapure water.

[0025] The formula for 200mL LB (Amp+) liquid culture medium is: 2g tryptone, 1g yeast extract, 2g sodium chloride, 20mg ampicillin, and 200mL ultrapure water.

[0026] (2) Further construction of recombinant baculovirus plasmids (Bacmid plasmids): 300 ng of each of the three different plasmids obtained above, HSV-2 gB2pFastBac1, HSV-2 gC2 pFastBac1, and HSV-2 gD2 pFastBac1, were transfected into 50 μL of DH10Bac competent cells (MAX Efficiency™ DH 10 Bac™ (ThermoFisher: 2498470A)) containing the Bacmid plasmid. Utilizing the characteristic of the Bacmid plasmid to homologously recombine exogenous genes into its own plasmid via the Mini-Tn7 transposon, the HSV-2 gB2, HSV-2 gC2, and HSV-2 gD2 gene sequences on the pFastBac1 plasmid were recombined and integrated into the Bacmid plasmid, resulting in three recombinant baculovirus plasmids containing different target gene sequences, namely HSV-2 Bacmid gB2, HSV-2 Bacmid pFastBac1, and HSV-2 gD2 pFastBac1. gC2, HSV-2 Bacmid gD2. The operation steps are as follows.

[0027] Before formally transfecting HSV-2 gB2 pFastBac1, HSV-2 gC2 pFastBac1, and HSV-2 gD2 pFastBac1 plasmids into DH10Bac competent cells, it is necessary to prepare the required LB selective agar plates for blue-white spot selection and preheat the cooled LB selective agar plates to 37°C in an incubator before use. Take 300 ng of each of the HSV-2 gB2 pFastBac1, HSV-2 gC2 pFastBac1, and HSV-2 gD2 pFastBac1 plasmids prepared in (1) above and add them to 50 μL of DH10Bac competent cells and mix gently. After incubating on ice for 30 min, heat shock at 42°C for 45 s, quickly transfer to ice and incubate on ice for another 2 min. Then add 950 μL of antibiotic-free SOC medium (Solepro: L1020-1L) and shake at 37°C and 225 rpm for 4 h to recover the bacteria. After the procedure, the recovered bacterial culture was cultured in antibiotic-free SOC medium for 10 minutes. -1 10 -2 10 -3Serial dilutions were performed, with 100 μL of each dilution transferred to preheated LB selective agar plates at 37°C. The plates were spread evenly and incubated upside down at 37°C for 48 hours. Blue and white colonies appeared at this point. White, independently growing positive colonies were picked and streaked onto LB selective agar plates to ensure all newly grown colonies were white positive. Single colonies were then inoculated into 10 mL of LB (TC+, K+, GM+) liquid medium (200 mL liquid medium formulation: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 200 mL ultrapure water, 50 µg / mL kanamycin, 7 µg / mL gentamicin, 10 µg / mL tetracycline). The medium was incubated at 37°C and 250 rpm for 8 hours. 1 mL of the bacterial culture was sent to a sequencing company for sequencing identification. 10 μL of the remaining bacterial culture was inoculated into 50 mL of... In LB (TC+,K+,GM+) liquid medium, the culture was carried out at 37℃ and 250rpm for 16h. Plasmids were extracted using the Beyotime brand baculovirus shuttle vector Bacmid small-scale extraction kit (Beyotime: D0031) to obtain successfully constructed recombinant HSV-2 Bacmid gB2 plasmid, recombinant HSV-2 Bacmid gC2 plasmid, and recombinant HSV-2 Bacmid gD2 plasmid.

[0028] The 200mL LB selective agar plate formulation includes: 2g tryptone, 1g yeast extract, 2g sodium chloride, 4g agar powder, 200mL ultrapure water / 50µg / mL kanamycin, 7µg / mL gentamicin, 10µg / mL tetracycline, 100µg / mL X-gal, and 40µg / mL IPTG.

[0029] 200mL LB (TC+, K+, GM+) liquid culture medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 200mL ultrapure water, 50µg / mL kanamycin, 7µg / mL gentamicin, 10µg / mL tetracycline.

[0030] 3. Expression and purification of the target product: Sf9 cells were cultured in Sf-900 II SFM medium (Thermo Fisher: 10902-096) on a shaker at 27°C and 125 rpm. When the cells reached the logarithmic growth phase (7 × 10⁶ cells / year), the culture time was determined. 6 / mL~8×10 6 At / mL, the cells were diluted to 1×10⁻⁶ cells with Sf-900 II SFM medium one day before transfection. 6 / 3mL was inoculated into 6-well plates. On the day of transfection, 10μL / well of ExpiFectamine SfTm transfection reagent (Thermo Fisher: A38915) was added to 250μL / well of Opti-MEM™ I serum-depleted medium (Gibco: 31985070) for dilution, and the mixture was inverted and incubated at room temperature for 5 min. Then, 1μg / well of recombinant HSV-2 Bacmid gB2 plasmid, recombinant HSV-2 Bacmid gC2 plasmid, and recombinant HSV-2 Bacmid gD2 plasmid were added to the three diluted transfection reagents, and the mixture was inverted and incubated at room temperature for 5 min. Finally, the above mixture was added to the Sf9 cells prepared the day before and cultured at 27℃ for 72-96 h until the Sf9 cells showed obvious pathological changes such as rounding, vacuolation, and budding. Then, the supernatant was collected after centrifugation at 500g / min for 5 min at 4℃ as the P1 generation recombinant baculovirus. The P1 generation virus was then passaged in Sf9 cells until the P3 generation recombinant baculovirus was obtained. Protein expression was detected using Western blot.

[0031] Protein expression was performed on 4L Sf9 cells infected with P3 generation virus. Cells were cultured in Sf-900 II SFM medium for 3 days and then harvested for subsequent purification. Protein expression was detected using Western blot. After centrifugation, the supernatant from the harvested cell samples was collected and incubated with Ni-NTA column material to obtain the target protein. The eluted samples from the first purification step were combined for a second step of Superdex 200 purification. The high-purity eluted samples were then concentrated and sterilely filtered through a 0.22 μm filter membrane. The final protein purity was analyzed using SDS-PAGE and Western blot. The primary antibody used for Western blot was Mouse-anti-His mAb (Proteintech: 66005-1-Ig). Protein concentration was detected using the Bradford method (BSA as a control). Finally, HSV-2 gB2 enveloped glycoprotein, HSV-2 gC2 enveloped glycoprotein and HSV-2 gD2 enveloped glycoprotein with a purity of over 90% were obtained. The purified proteins were dissolved in protein storage buffer and stored at -80℃.

[0032] Protein storage buffer formulation: 50 Mm Tris-HCl, 500 Mm NaCl, 5% (v / v) glycerol, pH 8.0.

[0033] 4. Immunogenicity evaluation of HSV-2 virus vaccine: To detect the immunogenicity of the vaccine of this invention, Balb / C mice were immunized with a mixture of the purified HSV-2 gB2 envelope glycoprotein, HSV-2 gC2 envelope glycoprotein, and HSV-2 gD2 envelope glycoprotein and adjuvant. The immunized Balb / C mice were purchased from the National Medical Primate Research Center; all were female, 6-8 weeks old, and weighed 18-25g. The mouse immunization experimental groups were set as follows: CpG / Alum group; gC2, gD2, gE2 group; and gB2, gC2, gD2 group. The CpG / Alum group served as a negative control for background control of the CpG / Alum adjuvant; the gC2, gD2, gE2 group served as a positive control for immunogenicity comparison with the gB2, gC2, gD2 group of this invention.

[0034] The immunization dose is calculated per mouse per injection as an example: The gB2, gC2, and gD2 groups of this invention consist of: 5 μg of HSV-2 gB2 enveloped glycoprotein, 5 μg of HSV-2 gC2 enveloped glycoprotein, 5 μg of HSV-2 gD2 enveloped glycoprotein, 50 μg of CpG, and 125 μg of Alum. Positive control group gC2, gD2, gE2 group: HSV-2 gC2 envelope glycoprotein 5μg, HSV-2 gD2 protein 5μg, HSV-2 gE2 envelope glycoprotein 5μg, CpG 50μg, Alum 125μg; Negative control group CpG / Alum group: CpG 50μg, Alum 125μg.

[0035] Eight mice were placed in each group. All mice were immunized via intramuscular injection into the posterolateral aspect of the thigh, receiving three doses of the vaccine on days 0, 14, and 28. Mice were sacrificed by cervical dislocation on day 42, and serum and spleen lymphocytes were collected for immunogenicity evaluation.

[0036] (1) Detection of specific antibody IgG: To determine the level of specific IgG antibodies in mouse serum after three doses of vaccine immunization, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of specific IgG antibodies against gB2, gC2, gD2, and gE2 proteins in mouse serum.

[0037] The purified antigen proteins HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2 gE2 were diluted to 1 μg / mL with ELISA coating buffer (Solepro: C1050). 100 μL of the diluted protein solution was added to each well of the coated plate, and the plate was incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were washed twice with PBST, patted dry, and then blocked at 37°C for 2 h with 200 μL of PBS containing 2% BSA (goat serum) (i.e., 2% BSA, obtained by dissolving 10 g of BSA powder in 500 mL of PBS). During the blocking interval, mouse serum was diluted with 2% BSA (goat serum) PBS in 96-well plates, starting with a 1:500 dilution and then serially diluted 3-fold. After blocking, discard the blocking solution, wash twice with PBST, pat dry, and add 100 μL of diluted mouse serum from the 96-well plate to the corresponding well. Incubate at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, and simultaneously dilute HRP-conjugated goat anti-mouse secondary antibody (Abcam: ab6789) at a 1:100000 ratio with 2% BSA (goat serum) in PBS, adding 100 μL to each well. Incubate at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, add 100 μL of TMB two-component chromogenic solution (Solepro: PR12102*50), react for 5-10 minutes, and stop the chromogenic reaction with 50 μL of stop solution (2M H2SO4) (Solepro: C1058). Read the absorbance (OD) value at 450 nm using a microplate reader. The minimum OD value that is greater than or equal to 2.1 times the OD value of the blank control well is used as the maximum dilution factor that can be detected by the serum.

[0038] The results are as follows Figure 1 As shown, the gB2, gC2, and gD2 groups of this invention induced mice to produce high levels of specific antibody IgG, reaching 1×10⁻⁶. 6 The antibody levels produced by the gB2, gC2, and gE2 groups are close to those of the positive control groups, suggesting that the gB2, gC2, and gD2 groups are no less effective than the currently reported positive control groups gC2, gD2, and gE2 groups in inducing mice to produce specific IgG antibodies. This vaccine has excellent application prospects and development value.

[0039] (2) Detection of specific antibody IgG subtypes IgG1 and IgG2a: To assess the levels of specific IgG1 and IgG2 induced in mice by the novel triantigen HSV-2 subunit vaccine, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of specific IgG1 and IgG2a antibodies in mouse serum. The experimental procedures are as follows: The purified antigen proteins HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2 gE2 were diluted with ELISA coating buffer (Solepro: C1050) at a concentration of 1 μg / mL for each protein to prepare a mixed protein solution containing all four proteins at a concentration of 1 μg / mL. 100 μL / well of the diluted mixed antigen protein solution was added to each well of the coated plate and incubated overnight at 4°C. The next day, the coating solution was discarded, the plate was washed twice with PBST, dried, and blocked with 200 μL / well of 2% BSA (goat serum) PBS at 37°C for 2 h. During the blocking interval, mouse serum was diluted with 2% BSA PBS in 96-well plates, starting with a 1:500 dilution and then serially diluted 3-fold. After blocking, discard the blocking solution, wash twice with PBST, pat dry, and add 100 μL of diluted mouse serum from the 96-well plate to the corresponding well. Incubate at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, and simultaneously dilute HRP-conjugated goat anti-mouse secondary antibodies IgG1 (abmam: ab97240) and IgG2a (abcam: ab97245) at a ratio of 1:100000 with 2% BSA (goat serum). Detect these antibodies separately, i.e., detect specific IgG1 and IgG2a in mouse serum in two separate plates. The dosage of the diluted secondary antibody is 100 μL per well, and incubate at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, add 100 μL of TMB two-component chromogenic solution (Solepro: PR12102*50), react for 5-10 min, and terminate the chromogenic reaction with 50 μL of stop solution (2M H2SO4) (Solepro: C1058). Read the absorbance (OD) value at 450 nm using a microplate reader. The lowest OD value greater than or equal to 2.1 times the OD value of the blank control well is taken as the maximum dilution factor detectable by this serum.

[0040] The results are as follows Figure 2 As shown, the overall level of specific IgG2a antibody in the serum of all mice was higher than that of IgG1, but the difference was not significant. Even though there were significant differences in the specific IgG1 results between the gB2, gC2, gD2 group of this invention and the positive control group gC2, gD2, gE group, the differences were not substantial. However, there was no significant difference in specific IgG2a, indicating that the immune responses induced by both groups were similar, that is, they tended to induce a relatively balanced Th1 / Th2 immune response.

[0041] (3) Serum neutralizing antibody titer determination: Since HSV-1 and HSV-2 belong to the same family of herpes simplex virus, they have high homology at the gene level and in amino acid sequence, and have the same way of entering the human body and the same latency mechanism. Therefore, neutralizing antibody tests were performed on HSV-1 and HSV-2 viruses respectively to evaluate the neutralizing ability of the gB2, gC2 and gD2 groups of the HSV-2 virus vaccine of the present invention against HSV-1 strain 17 (Strain17) and HSV-2 strain G (Strain G).

[0042] Two weeks after immunization with the three-dose vaccine, mouse serum was collected and frozen at -80°C. After thawing at room temperature, the serum was inactivated by incubating in a 56°C water bath for 30 min. The serum was then diluted in 96-well plates using DMEM medium (Vivacell: C3113-0500) at an initial dilution of 1:20, followed by 2-fold serial dilutions for a total of eight dilutions. Each sample was run in two replicates. Similarly, HSV-1 and HSV-2 viruses were diluted to 100 TCID50 per 50 μL using DMEM medium. The diluted serum and virus were then mixed at a 1:1 volume ratio in 96-well plates. A back-tipping experiment was also performed, with the virus solution (without serum at a concentration of 100 TCID50 per 50 μL) diluted 10-fold, 100-fold, and 1000-fold, with eight replicates for each dilution, to determine the viral titer at 100 TCID50 per 50 μL. Incubate at 37°C for 1 hour; during the waiting period, pre-passaged Vero cells with a confluence of over 90% are washed twice with PBS; then, digest with 0.25% trypsin for 2 minutes, and immediately add DMEM medium containing 4% FBS (Vivacell: C04001-050) and 2% penicillin-streptomycin (to stop digestion, resuspend the cells, count the cells, and adjust the cell suspension to 4×10⁶ cells / mL). 5 Cells / mL. Cell suspension was added at 100 μL / well to a serum-virus mixture incubated at 37°C for 1 h, and cultured in a 37°C, 5% CO2 cell culture incubator. Pathogenesis was observed after 6 days. The serum dilution corresponding to 50% cytopathic effect was the neutralizing titer of that serum. The DMEM medium containing 4% FBS (Vivacell: C04001-050) and 2% penicillin-streptomycin was prepared by adding 20 mL of FBS and 10 mL of penicillin-streptomycin to 470 mL of DMEM.

[0043] The results are as follows Figure 3As shown, the neutralizing titers of the gB2, gC2, and gD2 groups against both HSV-1 strain 17 and HSV-2 strain G were significantly higher than those of the negative control group CpG / Alum. Furthermore, the gB2, gC2, and gD2 groups exhibited neutralizing capabilities comparable to the positive control group gC2, gD2, and gE against HSV-2 strain G. Although the differences were significant, this demonstrates the effectiveness of the gB2, gC2, and gD2 groups in neutralizing HSV-2 strain G. In addition, in the cross-neutralization experiment against HSV-1, although the titers of the gB2, gC2, and gD2 groups were slightly lower than those of the positive control group gC2, gD2, and gE2, they still demonstrated neutralizing capabilities against HSV-1. This is highly advantageous for the subsequent development of vaccines that can simultaneously prevent both HSV-2 and HSV-1 viruses. Based on the combined neutralization results of the gB2, gC2, and gD2 groups against HSV-1 and HSV-2 viruses, this indicates that the gB2, gC2, and gD2 groups of this invention have good effects in preventing HSV-1 and HSV-2, and are expected to be developed into a novel vaccine that effectively prevents HSV-2 and provides cross-protection against HSV-1.

[0044] (4) Detection of cellular immune response: To evaluate the level of specific T cell response induced by the vaccine of this invention, we used an enzyme-linked immunospot (ELISPOT) assay to detect the number of T cells that specifically secrete IFN-γ and IL-4 cytokines per million spleen lymphocytes, and thus judged the T cell response by the number of positive cells.

[0045] Two weeks after immunization with three doses of vaccine, mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol solution for 30 seconds, and their spleens were removed under aseptic conditions. A 70µm pore size cell sieve was placed in agar plates, and 4mL of serum-free RPMI 1640 medium (Vivacell: 2441385) was added. The spleen was ground using a syringe plunger, and cells were filtered through the sieve until residual white connective tissue remained. Single cells from the spleen were transferred to 15mL centrifuge tubes. The tubes were centrifuged at 500g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended. 2mL of erythrocyte lysis buffer (Solepro: R1010) was added, and the reaction was stopped after 4 minutes by adding 10mL of RPMI 1640 containing 10% FBS. The tubes were centrifuged at 500g for 10 minutes at 4°C, the supernatant was discarded, and the pellet was resuspended. 1mL of RPMI 1640 containing 10% FBS was added and mixed thoroughly. Cell counting was then performed, and the cell concentration was adjusted to 1×10⁻⁶. 7cells / mL. Following the instructions of the Mabtech ELISpot Plus: Mouse IFN-γ (ALP) (Mabtech: 3321-4AST-2) and ELISpot Plus: Mouse IL-4 (ALP) (Mabtech: 3311-4APW-2) kits, prepare the previously prepared splenocytes at a density of 250,000 per well, and add them to each well containing four of the same concentrations of HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2... The gE2 protein mixed antigen protein solution was mixed with the cell solution at a working concentration of 2 μg / mL in a 1:1 volume ratio in a 96-well plate. 200 μL of the resulting cell suspension was slowly pipetted into the wells along the sidewall of the ELISPOT plate. Two stimulation wells and one blank background well were prepared for each sample. Three positive control wells were also included, stimulated with 1×PMA (Lianke Biotechnology: CS1001). All plates were then incubated at 37°C and 5% CO2 for 36 hours, ensuring sterility and avoiding plate movement. The plates were then washed and incubated with antibodies according to the kit instructions. After staining and air-drying in the dark, cell counts were performed using a fluorescent ELISA spectrophotometer. The number of spots in the stimulation wells was subtracted from the number of spots in the blank background wells to remove background interference. The resulting data were summarized, normalized, and analyzed to calculate the number of specific T cells secreting IFN-γ and IL-4 per million spleen cells. The method for preparing RPMI 1640 medium containing 10% FBS is to add 50 mL of FBS to 450 mL of RPMI 1640 medium.

[0046] The results are as follows Figure 4 As shown, compared with the negative control group CpG / Alum group, the gB2, gC2, and gD2 groups of the present invention induced higher levels of IFN-γ+ and IL-4+ specific T cell responses. Among them, the IFN-γ+ specific T cell response level in the gB2, gC2, and gD2 groups was comparable to that in the positive control group gC2, gD2, and gE2 groups. Importantly, the IL-4+ specific T cell response in the gB2, gC2, and gD2 groups was slightly better than that in the gC2, gD2, and gE2 groups. Although there was no significant difference, it still indicates that the gB2, gC2, and gD2 groups have the potential to induce IL-4+ specific T cell responses.

[0047] (5) Detection of secreted cytokines: To evaluate the level of immune response in mice induced by the novel triantigen HSV-2 subunit vaccine in the Th1 and Th2 directions, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of Th1 cytokines IFN-γ and IL-2 and Th2 cytokines IL-4 and IL-10 in the culture supernatant of spleen lymphocytes.

[0048] The peripheral spleen lymphocytes prepared in (4) above were seeded into 24-well cell culture plates, with 1 × 10⁶ cells per well. 7 Cells were cultured in 1 mL volume, and a mixed antigen protein stimulant containing four HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2 gE2 proteins at the same concentration was added to each well. The working concentration of the mixed antigen protein stimulant was 2 μg / mL. A positive control well was also prepared, containing concanavalin A (Solepro: IC4870-25 mg) at a working concentration of 10 μg / mL. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h, followed by centrifugation at 4°C and 1000 rpm for 10 min to collect the supernatant. The concentrations of cytokines IFN-γ, IL-2, IL-4, and IL-10 in the spleen lymphocyte culture supernatant were detected using the Thermo Fisher Mouse Th1 Th2 Uncoated ELISA kit (Thermo Fisher: 88-7711-44).

[0049] The results are as follows Figure 5 As shown, the gB2, gC2, and gD2 groups of this invention induced mouse splenic lymphocytes to produce higher concentrations of IFN-γ and IL-2 cytokines, with concentration levels comparable to the positive control groups gC2, gD2, and gE2. Regarding the concentration of IL-4 cytokine, the gB2, gC2, and gD2 groups of this invention were even superior to the positive control groups gC2, gD2, and gE. However, in the IL-10 detection results, the concentrations of cytokines in the cell culture supernatant of both groups were comparable and showed no significant difference.

[0050] Based on the above experimental data, the results indicate that the gB2, gC2, and gD2 combination shows good potential in preventing HSV-2 and also has a neutralizing effect on HSV-1. Among the numerous HSV-2 vaccines studied previously, this combination is reported for the first time. In addition to gB2 effectively inducing humoral immunity in mice, the gC2 and gD2 antigens are added. Since these three envelope proteins are mainly involved in HSV-2 entry into the host and immune escape, selecting these three envelope proteins to prepare an HSV-2 vaccine is of great significance. This vaccine combination not only broadens the path of HSV-2 vaccine development but also provides an effective and safe candidate vaccine for HSV-2 prevention, possessing significant application value.

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A novel triantigenic HSV-2 subunit vaccine, characterized in that, The antigen comprises HSV-2 gB2 envelope glycoprotein, HSV-2 gC2 envelope glycoprotein and HSV-2 gD2 envelope glycoprotein, and the complex adjuvant is CpG oligonucleotide and aluminum adjuvant. The amino acid sequence of the HSV-2 gB2 envelope glycoprotein is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO.

4. The amino acid sequence of the HSV-2 gC2 envelope glycoprotein is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO.

5. The amino acid sequence of the HSV-2 gD2 envelope glycoprotein is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO.

6.

2. The novel triantigenic HSV-2 subunit vaccine according to claim 1, characterized in that, The mass ratio of the HSV-2 gB2 envelope glycoprotein, the HSV-2 gC2 envelope glycoprotein, the HSV-2 gD2 envelope glycoprotein, the CpG oligonucleotide and the aluminum adjuvant is 5:5:5:50:

125.

3. A method for preparing a novel triantigenic HSV-2 subunit vaccine according to claim 1 or 2, characterized in that, The method comprises the following steps: The HSV-2 gB2 envelope glycoprotein, the HSV-2 gC2 envelope glycoprotein and the HSV-2 gD2 envelope glycoprotein dissolved in the protein storage buffer are mixed with the aluminum adjuvant, and then placed at 4°C for slow shaking overnight, and then mixed with the CpG oligonucleotide before vaccination, so as to obtain the novel three-antigen HSV-2 subunit vaccine.

4. The process for the preparation of novel tri-antigenic HSV-2 subunit vaccine as claimed in claim 3, wherein, The protein storage buffer comprises 50Mm Tris-HCI, 500Mm NaCI and 5% glycerol, and has a pH of 8.

0.

5. The process for the preparation of novel tri-antigenic HSV-2 subunit vaccine as claimed in claim 3, wherein, The shaking speed is 80rpm / min.

6. The novel three-antigen HSV-2 subunit vaccine of claim 1 or 2 is used for preparing a medicine for preventing and / or treating HSV-2.

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