Hsv-2 triantigenic subunit vaccine and methods of making and using same

Abstract

The present application relates to HSV-2 three antigen subunit vaccine and its preparation method and application, belong to the field of biotechnology. The vaccine comprises HSV-2 recombinant protein and composite adjuvant;HSV-2 recombinant protein includes the extracellular domain of three proteins of HSV-2 gB2, HSV-2 gD2, HSV-2 gE2;Composite adjuvant is composed of CpG oligodeoxynucleotide and aluminum adjuvant. The HSV-2 three antigen subunit vaccine of the present application takes HSV-2 gB2, gD2, gE2 protein as antigen, takes CpG oligodeoxynucleotide and aluminum adjuvant as composite adjuvant, immunizes BALB / c mouse by the way of intramuscular injection, successfully induces high level gB2, gD2, gE2 specific IgG antibody, and effectively improves the neutralization ability of immune serum to HSV-2 virus strain and the cross neutralization ability to HSV-1 virus strain.

Description

Technical Field

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

[0002] Herpes simplex virus type 2 (HSV-2) belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, and Herpes simplex virus genus. It is an enveloped double-stranded DNA virus with a genome length of approximately 152 kb, composed of two independent and covalently linked segments, UL (unique long) and US (unique short), encoding approximately 85 open reading frames. Viral replication involves the production of various viral assembly-related proteins, beginning with immediate-early (IE) proteins, followed by early (E) proteins and late (L) structural proteins. HSV-2 requires viral fusion with the cell membrane to enter target cells. Currently, 12 HSV-2 envelope glycoproteins have been identified, including gB-gN, of which four (gD, gB, gH, and gL) are involved in viral entry into host cells. First, gB and gC interact with the cell surface receptor heparan sulfate proteoglycan (HSPG), allowing the virus to attach to the host cell surface; this process facilitates subsequent viral entry. Located on the surface of the viral particle, gD exists in monomeric form with a closed conformation. Its extracellular domain's C-term region encloses an unstructured N-term region, presenting an inactive pre-fusion conformation. When gD binds to cell surface receptors, it adopts an activated open conformation, where the C-terminus of its extracellular domain moves away from the N-terminus, causing the latter to structure and form a hairpin. Subsequently, gD binds to cell surface receptors such as herpesvirus entry mediator (HVEM, I) or immunoglobulin superfamily members nectin-1, nectin-2 (II) or 3-O-sulfotransferase (III). Finally, gB and the heterodimer gH / gL jointly mediate the fusion of the viral envelope with the host cell membrane, thereby injecting the decapitated viral capsid into the host cell. The capsid is then transported to the nucleus along microtubules and, through interaction with the nuclear pore complex, squeezes the viral genome into the nucleus for progeny viral replication.

[0003] Herpes simplex virus enters the body through tiny cracks in the skin or mucous membranes, usually through direct skin contact or sexual contact. The virus invades local epithelial cells, primarily in the epidermis or mucous membranes of the genital area. The infection that causes lesions in the host cell and surrounding cells upon initial contact with the virus is called primary lysis infection. Subsequently, the viral genomic DNA can be retrogradely transported via cell axons to sensory neurons in the lumbosacral dorsal root ganglia. These cells express only low levels of HLA molecules and the virus expresses only a small amount or no protein or RNA transcripts. At this stage, the infected host shows no pathological symptoms. However, when the latent virus is stimulated by external factors such as immune-related stimuli, physical and chemical stimuli (e.g., ultraviolet radiation), menstruation, stress, and traumatic events, the virus will be transported from the latent site along peripheral neurons up the axon to the mucous membranes or epithelial cells, causing a re-emergence infection. This pattern of infection is called latent recurrent infection. This phenomenon occurs due to immune escape, whereby the herpesvirus downregulates the host's immune response through various means, including infecting dendritic cells and various immune cells, continuously refining its infection strategy and gradually adapting to the surveillance of the host's immune system to establish an effective infection. In oral-facial infections caused by HSV-1, the virus establishes latency in the trigeminal ganglion, while in genital infections caused by HSV-2, the virus remains latent in pelvic visceral neurons.

[0004] Currently, antiviral drugs are the only treatment approved by the U.S. Food and Drug Administration (FDA) for treating herpes virus infections. Treatments include acyclovir, but because viral infections are often accompanied by asymptomatic shedding or mild itching, patients are unaware of their infection, making HSV-2 prevention extremely difficult. Furthermore, long-term use of antiviral drugs can easily lead to the development of drug-resistant mutant strains. Therefore, developing an HSV-2 vaccine is the most economical and effective means of preventing and controlling the virus. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies and provide an HSV-2 triantigen subunit vaccine, its preparation method, and its application. By evaluating its immunogenicity in mice, the feasibility of using it as a vaccine for the prevention and / or treatment of HSV-2 is verified, which is expected to provide theoretical support for the development of HSV-2 vaccines.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides an HSV-2 triantigen subunit vaccine, the vaccine comprising HSV-2 recombinant protein and a complex adjuvant; the HSV-2 recombinant protein comprises extracellular domains of three proteins: HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2, wherein the amino acid sequences of the extracellular domains of HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the complex adjuvant is composed of CpG oligodeoxynucleotides and aluminum adjuvant.

[0008] Furthermore, the mass ratio of the extracellular domain of HSV-2 gB2 protein, the extracellular domain of HSV-2 gD2 protein, the extracellular domain of HSV-2 gE2 protein, CpG oligonucleotide, and aluminum adjuvant is 5:5:5:50:125.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned HSV-2 triantigen subunit vaccine, comprising the following steps:

[0010] Step (1) The coding gene sequences corresponding to the extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 were cloned into the HindIII and EcoRI restriction sites of the pFastBac1 plasmid to construct recombinant pFastBac1gB2 plasmid, recombinant pFastBac1gD2 plasmid, and recombinant pFastBac1gE2 plasmid;

[0011] Step (2): Obtain the target fragments of the three protein-coding genes from the recombinant pFastBac1gB2 plasmid, the recombinant pFastBac1gD2 plasmid, and the recombinant pFastBac1gE2 plasmid, respectively, and insert them into the Bacmid plasmid to construct the three recombinant Bacmid plasmids.

[0012] Step (3): The three recombinant Bacmid plasmids constructed in step (2) were expressed by the eukaryotic expression system sf9 cells. The proteins were purified by nickel column purification to obtain the extracellular domains of HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 respectively.

[0013] Step (4): The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 obtained in step (3) are dissolved in phosphate buffer and a compound adjuvant is added to it before immunization to prepare HSV-2 triantigen subunit vaccine.

[0014] Step (4): The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 obtained in step (3) are dissolved in phosphate buffer and a compound adjuvant is added to it before immunization to prepare HSV-2 triantigen subunit vaccine.

[0015] Further, 5 μg of each of the three extracellular domains of HSV-2 gB2, gD2 and gE2 proteins obtained in step (3) were dissolved in phosphate buffer to prepare 15 μg / injection; before immunization, 50 μg / injection of compound adjuvant CpG oligodeoxynucleotide and 125 μg / injection of aluminum adjuvant were added to prepare HSV-2 triantigen subunit vaccine.

[0016] A third aspect of the present invention provides the use of the above-mentioned HSV-2 triantigen subunit vaccine in the preparation of a vaccine for the prevention or treatment of diseases caused by herpes simplex virus infection.

[0017] Furthermore, herpes simplex virus includes herpes simplex virus type 1 and herpes simplex virus type 2.

[0018] In this invention, the concentration of the extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 in the vaccine is 5 μg / injection, the amount of CpG oligodeoxynucleotide added is 50 μg / injection, and the amount of aluminum adjuvant added is 125 μg / injection.

[0019] In this invention, the vaccine is administered via intramuscular injection.

[0020] In this invention, the vaccine is a cross-immunization protection vaccine against HSV-2 and HSV-1 virus strains.

[0021] In this invention, the vaccine composition can effectively enhance the neutralizing capacity of immune serum against HSV-2 virus strains and its cross-neutralizing capacity against HSV-1 virus strains.

[0022] This invention demonstrates through comparative experiments using three different combinations of antigens that the humoral and cellular immune responses induced by the subunit vaccine using the gB2, gD2, and gE2 triantigens of this invention are comparable to those induced by the previously proven effective gC2, gD2, and gE2 triantigen vaccines. Furthermore, the neutralizing effect of mouse serum after vaccination with the gB2, gD2, and gE2 triantigen subunit vaccine is comparable to that of mouse serum in the gC2, gD2, and gE2 triantigen group, laying the foundation for the widespread adoption of subunit vaccines.

[0023] Compared with the prior art, the beneficial effects of this invention are as follows:

[0024] 1. The HSV-2 triantigen subunit vaccine of the present invention uses HSV-2 gB2, gD2, and gE2 proteins as antigens and CpG oligodeoxynucleotides and aluminum adjuvants as compound adjuvants. It is administered to BALB / c mice via intramuscular injection, successfully inducing high levels of gB2, gD2, and gE2 specific IgG antibodies, and effectively improving the neutralizing capacity of immune serum against HSV-2 (Strain G) virus strain and cross-neutralizing capacity against HSV-1 (Strain17) virus strain.

[0025] 2. The HSV-2 triantigen subunit vaccine of this invention induces a strong Th1-type CD4+ activating response. + Antigen-specific cellular immune responses, primarily T-cell-based.

[0026] 3. Comparative experiments revealed that the HSV-2 triantigen (gB2, gD2, gE2) subunit vaccine of the present invention has a comparable level of immunogenicity evaluation to the previously proven effective triantigen vaccine (gC2, gD2, gE2). Attached Figure Description

[0027] Figure 1 The results of mouse serum ELISA detection in this invention are shown below; where A: HSV-2 specific IgG antibody detection; B: HSV-2 specific IgG1 and IgG2a antibody detection; C: HSV-2 specific IgG1 and IgG2a antibody ratio; Blank is the blank control well.

[0028] Figure 2 The results of the mouse serum neutralization experiment of this invention are shown; where A: neutralizing antibody titer of HSV-2 G strain; B: neutralizing antibody titer of HSV-1 17 strain;

[0029] Figure 3 The results of ELISA detection of cytokines in this invention are as follows: A: Concentration of Th1-type (IFN-γ, IL-2) cytokines in spleen cell supernatant; B: Concentration of Th2-type (IL-4, IL-10) cytokines in spleen cell supernatant.

[0030] Figure 4 The following are Elispot results from this invention: A: Number of IFN-γ spots; B: Number of IL-4 spots; C: Representative photograph of the Elispot reaction of IFN-γ; D: Representative photograph of the Elispot reaction of IL-4. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the embodiments.

[0032] 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.

[0033] Example 1: Preparation of experimental HSV-2 gB2, gD2, gE2 triantigen subunit vaccine

[0034] An HSV-2 triantigen subunit vaccine, the vaccine comprising HSV-2 recombinant protein and a complex adjuvant; the HSV-2 recombinant protein comprising extracellular domains of three proteins, gB2, gD2, and gE2, the amino acid sequences of the extracellular domains of HSV-2 gB2, gD2, and gE2 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the complex adjuvant is composed of CpG oligodeoxynucleotides and aluminum adjuvant.

[0035] The amino acid sequence design scheme for the extracellular domain of HSV-2 gB2 is: GP67 Signal peptide + gB2 (98-730aa) + His-tag + stop codon. The amino acid sequence of the extracellular domain of HSV-2 gB2 is shown in SEQ ID NO.1.

[0036] The amino acid sequence design scheme for the extracellular domain of HSV-2 gD2 is: GP67 Signal peptide + gD2 (26-310aa) + His-tag + stop codon. The amino acid sequence of the extracellular domain of HSV-2 gD2 is shown in SEQ ID NO.2.

[0037] The amino acid sequence design scheme for the extracellular domain of HSV-2 gE2 is: GP67 Signal peptide + gE2 (21-414aa) + His-tag + stop codon. The amino acid sequence of the extracellular domain of HSV-2 gE2 is shown in SEQ ID NO.3.

[0038] The preparation method for the experimental HSV-2 triantigen subunit vaccine group is as follows:

[0039] Step (1), transformation of plasmids containing gB2, gD2, and gE2 gene expression:

[0040] The gene sequences encoding the extracellular domains of the three HSV-2 proteins gB2, gD2, and gE2 were cloned into the HindIII and EcoRI restriction sites of the pFastBac1 plasmid to construct a recombinant pFastBac1 plasmid. 400 ng of the three recombinant pFastBac1 plasmids were added to 100 μL of competent E. coli DH5α cells, mixed well, incubated on ice for 30 min, then heat-shocked at 42°C for 45 s, quickly transferred to ice and incubated on ice for another 2 min, then 500 μL of antibiotic-free SOC medium (Solepro: L1020-1L) was added, and the culture was shaken at 37°C and 120 rpm for 1 h. 100 μL of the bacterial culture was spread on a solid medium containing ampicillin and incubated upside down at 37°C for 12 h.

[0041] Single colonies were picked and inoculated into 10 mL of liquid medium containing ampicillin, 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 liquid medium containing ampicillin, and cultured at 37°C and 250 rpm for 16 h. Plasmids were extracted using the PureYield plasmid Maxiprep system plasmid extraction kit to obtain pFastBac1gB2, pFastBac1gD2, and pFastBac1gE2 plasmid DNA.

[0042] The formula for 200 mL of ampicillin-resistant solid culture medium is as follows: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 4 g agar powder, 20 mg ampicillin, and 200 mL water.

[0043] The formula for 200mL of ampicillin-resistant liquid culture medium is as follows: 200mL ampicillin liquid culture medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 20mg ampicillin, 200mL water.

[0044] Step (2), preparation of recombinant Bacmid DNA:

[0045] Recombinant pFastBac1gB2, pFastBac1gC2, and pFastBac1gD2 plasmids containing the target gene, which were correctly sequenced, were extracted and transformed into MAX Efficiency™ DH10Bac™ (Thermo Fisher: 2498470A) competent cells for transposition into Bacmid plasmids. Colonies containing recombinant Bacmid plasmids were screened and identified using blue-white screening.

[0046] The specific method for preparing recombinant Bacmid DNA is as follows:

[0047] Prepare solid selective culture media containing four different antibiotics in advance and preheat them to 37°C before use. Add 300 ng of recombinant pFastBac1gB2 plasmid DNA, recombinant pFastBac1gD2 plasmid DNA, and recombinant pFastBac1gE2 plasmid DNA to 50 μL of competent *E. coli* cells DH10Bac, mix gently, incubate 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 incubate at 37°C and 225 rpm for 4 h. Perform a 10-day recovery period with SOC medium (Solepro: L1020-1L). -1 10 -2 10 -3 Serial dilutions were performed, with 100 μL of each dilution added to preheated solid selective medium at 37°C. The culture was spread evenly and incubated upside down at 37°C for 48 hours. At this point, blue and white colonies appeared. Isolated white positive colonies were picked and streaked onto solid selective medium to ensure that all newly grown colonies were white positive. Single colonies were picked and inoculated into 10 mL of liquid selective medium (200 mL liquid selective medium: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 200 mL water). The culture was incubated at 37°C and 250 rpm for 8 hours. 1 mL of the culture was sent to a sequencing company for sequencing identification. The remaining culture was used to extract plasmids using a baculovirus shuttle vector Bacmid mini-extraction kit (Beyotime: D0031) to obtain gB2 recombinant Bacmid DNA, gD2 recombinant Bacmid DNA, and gE2 recombinant Bacmid DNA.

[0048] The formulation of 200 mL of solid selective medium containing four resistances is as follows: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 4 g agar powder, 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X-gal, 40 μg / mL IPTG, and 200 mL water.

[0049] The formula for 200 mL of solid selective medium is: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 4 g agar powder, 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X-gal, 40 μg / mL IPTG, and 200 mL water.

[0050] The formulation of 200 mL liquid selective culture medium is as follows: 2 g tryptone, 1 g yeast extract, 2 g sodium chloride, 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, and 200 mL water.

[0051] Step (3), viral amplification and purification:

[0052] Sf9 cells were cultured in Sf-900 II SFM medium (Thermo Fisher: 10902-096) at 27°C in Erlenmeyer flasks on a shaker at 125 rpm. When the cells reached the logarithmic growth phase (7-8 × 10⁶ cells / year),... 6 When the cell viability is >95%, the cells are injected at a rate of 1×10⁹ / mL. 6 Inoculate 2 mL of the solution into a 6-well plate and incubate for 15 min. Then, take 10 μL of ExpiFectamine Sf per well. Tm The transfection reagent (Thermo Fisher: A38915) was diluted in 250 μL / well of Opti-MEM™ I serum-depleted medium (Gibco: 31985070), mixed by inverting, and incubated at room temperature for 5 min. Then, 1 μg / well of gB2 recombinant Bacmid DNA, gD2 recombinant Bacmid DNA, and gE2 recombinant Bacmid DNA were added to the diluted transfection reagent, mixed by inverting, and incubated at room temperature for 5 min. Finally, the above mixture was added to sf9 cells and cultured at 27°C for 72-96 h until the sf9 cells showed obvious pathological phenomena such as rounding and swelling, budding, and breakage.

[0053] Centrifuge at 300×g for 5 min to collect the supernatant, which will be used as the P0 generation virus stock solution.

[0054] Then take 20-80 μL of P0 generation virus and inoculate it into 25 mL (1×10⁻⁶). 6 The P1 generation virus was amplified in sf9 cells ( / mL), and then the P1 generation was amplified to the P2 generation for subsequent infection.

[0055] P2 generation virus was used to infect 8L Sf9 cells with MOI=1 for protein expression. The cells were cultured in Sf-900 II SFM medium for 3 days and then harvested for subsequent purification. Protein expression was detected by Western blot.

[0056] After centrifugation, the supernatant from the harvested cell samples was collected and incubated using a nickel column to obtain the target protein. The eluted samples from the first-step purification were combined and subjected to a second-step Superdex 200 purification. The eluted samples with a purity of 90% were concentrated and then aseptically filtered through a 0.22 μm 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 (with BSA as a control).

[0057] Step (4), preparation of vaccine injection:

[0058] The purified HSV-2 gB2, gD2, and gE2 proteins, each 5 μg, were dissolved in phosphate buffer to prepare a 15 μg / injection dose. Before immunization, 50 μg / injection of CpG oligodeoxynucleotides and 125 μg / injection of aluminum adjuvant were added to the solution to prepare the HSV-2 gB2, gD2, and gE2 triantigen subunit vaccine.

[0059] Comparative Example 1: Preparation of Control Group for HSV-2 gC2, gD2, gE2 Triantigen Subunit Vaccine

[0060] A proven effective HSV-2 triantigen subunit vaccine comprising HSV-2 recombinant protein and a complex adjuvant; the HSV-2 recombinant protein includes extracellular domains of three proteins: gC2, gD2, and gE2, the amino acid sequences of the extracellular domains of HSV-2 gC2, gD2, and gE2 are shown in SEQ ID NO.4, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the nucleotide sequences are shown in SEQ ID NO.8, SEQ ID NO.6, and SEQ ID NO.7, respectively; the complex adjuvant is composed of CpG oligodeoxynucleotides and aluminum adjuvant.

[0061] The amino acid sequence design scheme for the extracellular domain of HSV-2 gC2 is: GP67 Signal peptide + gC2 (28-447aa) + His-tag + stop codon. The amino acid sequence of the extracellular domain of HSV-2 gC2 is shown in SEQ ID NO.4.

[0062] The preparation method for the HSV-2 gC2, gD2, gE2 triantigen subunit vaccine control group is the same as above.

[0063] Immunogenicity evaluation of the vaccine of this invention:

[0064] To test the immunogenicity of the vaccine, we immunized BALB / c mice with a mixture of purified protein and adjuvant. The BALB / c mice used were purchased from the National Medical Primate Research Center; all were female and 6-8 weeks old. Mouse immunization experiment setup:

[0065] gB2, gD2, gE2 experimental group: gB2, gD2, gE2 + CpG / Alum;

[0066] gC2, gD2, gE2 positive control group: gC2, gD2, gE2 + CpG / Alum;

[0067] Negative control group: CpG / Alum.

[0068] Mice were immunized with 5 μg / injection of each antigen protein, 50 μg / injection of CpG, and 125 μg / injection of aluminum adjuvant per injection. Eight mice were in each group, and the vaccines were administered via intramuscular injection into the posterolateral thigh. All mice received three immunizations on days 0, 14, and 28. Mice were sacrificed by cervical dislocation on day 42, and serum and splenic lymphocytes were collected for immunogenicity evaluation.

[0069] Specific antibody IgG detection:

[0070] 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.

[0071] The purified antigens gB2, gC2, gD2, and gE2 were diluted to 1 μg / mL with ELISA coating buffer (Solepro: C1050) and added to each well at 100 μL. The plates were then incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were washed twice with 1×PBST (Sevier: G2157-1L), patted dry, and then 200 μL / well of PBS containing 2% BSA (10 g of BSA powder dissolved in 500 mL of PBS) was added. The plates were blocked at 37°C for 2 h. Subsequently, mouse serum was diluted with PBS containing 2% BSA, starting at a 1:500 dilution and then serially diluted 3-fold to a final dilution of 1:88573500. After blocking, discard the blocking solution, wash twice with 1×PBST, pat dry, and add 100 μL of diluted mouse serum (1:500~1:88573500) to each well of the blocked plate. Also include two blank control wells with PBS. Incubate at 37°C for 1 h. After incubation, wash four times with 1×PBST, pat dry, and add 100 μL of the diluted HRP-conjugated goat anti-mouse secondary antibody (abcam: ab6789) to each well using PBS containing 2% BSA at a ratio of 1:100000. Incubate at 37°C for 1 h. After incubation, wash four times with 1×PBST, pat dry, and add 100 μL of TMB two-component chromogenic solution (Solepro: PR12102*50). React for 5-10 min, and stop the chromogenic reaction with 50 μL of stop solution (2M H2SO4) (Solepro: C1058). The OD450 value was read using a microplate reader. A value greater than 2.1 times that of the blank control was considered positive, and the antibody titer was the corresponding dilution factor. Results are as follows: Figure 1 As shown in Figure A, the vaccine of the present invention induced mice to produce high levels of specific antibodies IgG. The antibody levels produced by the gB2, gD2, and gE2 experimental groups were close to those of the gC2, gD2, and gE2 positive control groups. This suggests that the vaccine of the present invention is no less effective than the gC2, gD2, and gE2 positive control groups, which have better effects in the present invention, and has excellent application prospects and development value.

[0072] Detection of specific antibody IgG subtypes IgG1 and IgG2a:

[0073] To evaluate the IgG1 and IgG2a immune responses induced by the vaccine of this invention in mice, we used an enzyme-linked immunosorbent assay (ELISA) to detect the levels of specific IgG1 and IgG2a antibodies in mice. Except for the coating solution containing a mixed antigen of gB2, gC2, gD2, and gE2 (1 μg / mL of each antigen), the experimental procedures were the same as those for the specific antibody IgG detection experiment. IgG1 (abcam: ab97240), IgG2a (abcam: ab97245). The results are as follows: Figure 1 As shown in B and 1C, the overall IgG2a response level was higher than that of IgG1, but the difference was not significant. Furthermore, the responses of the gB2, gD2, and gE2 experimental groups and the gC2, gD2, and gE2 positive control group were at similar levels, indicating that the immune capabilities induced by both groups were similar. Both tended to promote a relatively balanced Th1 and Th2 response, that is, slightly tended to induce a relatively balanced humoral and cellular immune response. Balancing these two immune responses can effectively enhance the immunogenicity of the vaccine.

[0074] Serum neutralizing antibody titer assay:

[0075] To assess the ability of neutralizing antibody titers to block HSV-1 (Strain17) and HSV-2 (Strain G) viral infection, mouse serum was inactivated at 56°C for 30 min. The serum was then diluted with DMEM medium (Vivacell: C3113-0500) at an initial dilution of 1:20, followed by 2-fold serial dilutions up to a total of 8 dilutions up to 1:2560. Each sample was performed in duplicate. Similarly, HSV-1 and HSV-2 viruses were diluted to 100 TCID using DMEM medium. 50 50 μL of serum was added per well; then the diluted serum and virus were mixed at a volume ratio of 1:1 in a 96-well plate. A back-tipping experiment was also performed, with the virus solution diluted 10-fold, 100-fold, and 1000-fold, with eight replicates for each dilution, to determine the viral titer at 100 TCID50. 50 Incubate at 37°C for 1 h in 50 μL increments; wash Vero cells twice with PBS, then digest with 0.25% trypsin for 2 min, add DMEM containing 4% FBS (VivaCell: C04001-050) and 1% penicillin-streptomycin to terminate digestion, resuspend and count the cells, and adjust the cell suspension to 4 × 10⁶ cells / mL. 5 Cells / mL were added to the incubated serum-virus mixture at a rate of 100 μL / well 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 defined as the neutralizing titer of the 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.

[0076] The results are as follows Figure 2As shown, the neutralizing titers of each vaccine group were significantly higher than those of the CpG / Alum negative control group. Furthermore, the gB2, gD2, and gE2 experimental groups and the positive control group showed comparable neutralizing abilities against HSV-1 and HSV-2, indicating that this vaccine combination has good ability to prevent both HSV-1 and HSV-2 and is expected to be developed into an effective vaccine for preventing HSV-2.

[0077] Cytokine level detection:

[0078] To detect the mouse immune response to Th1 and Th2 cytokines induced by the vaccine of this invention, 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. Mouse spleens were removed under aseptic conditions. A 70 μm pore size cell sieve was placed in a culture dish, and 4 mL of RPMI 1640 medium (VivaCell: 2441385) was added. The spleen was ground using a syringe plunger, and cells were filtered through the sieve until only white connective tissue remained. Single cells from the spleen were transferred to 15 mL centrifuge tubes. The cells were centrifuged at 4°C and 500 × g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 2 mL of erythrocyte lysis buffer (Solepro: R1010). After reacting for 4 minutes, 10 mL of RPMI 1640 containing 10% FBS and 1% penicillin was added to terminate the reaction. The cells were centrifuged at 4°C and 500 × g for 10 minutes, and the supernatant was discarded. Add 1 mL of 10% FBS and 1% penicillin-dextrin antibody to RPMI 1640 and resuspend the pellet thoroughly. Perform spleen cell counting and adjust the cell concentration to 1×10⁻⁶. 7 / mL. Cells were seeded into 24-well cell culture plates, 1 mL per well, and gB2, gC2, gD2, and gE2 antigens were added to the wells for stimulation at a concentration of 2 μg / mL. Positive control wells were stimulated with 10 μg / mL concanavalin A (Solepro: IC4870-25 mg). The plates were incubated at 37°C in a 5% CO2 incubator for 48 h, followed by centrifugation at 1000 rpm for 10 min at 4°C 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). Results are shown below. Figure 3 As shown, the gB2, gD2, and gE2 experimental groups of the present invention induce mouse splenic lymphocytes to produce high levels of IFN-γ, IL-2, IL-4, and IL-10 cytokines, which are comparable to the levels of the gC2, gD2, and gE2 positive control groups, indicating that they can induce mice to produce a strong level of cellular immunity.

[0079] The preparation method for RPMI 1640 containing 10% FBS and 1% penicillin antibody is as follows: add 50 mL of FBS and 10 mL of penicillin antibody to 440 mL of RPMI 1640 medium.

[0080] Cellular immune response detection:

[0081] To evaluate the cellular immunity level of the vaccine of the present invention, the number of T cells in spleen lymphocytes that specifically secrete IFN-γ and IL-4 cytokines was detected by enzyme-linked immunospot (ELISpot) assay. According to the instructions of Mabtech's Elispot Plus: Mouse IFN-γ (ALP) (Mabtech: 3321-4AST-2) and Elispot Plus: Mouse IL-4 (ALP) (Mabtech: 3311-4APW-2) kits, prepare 250,000 pre-prepared spleen cell suspensions per well and mix them with gB2, gC2, gD2, and gE2 antigen stimulants at a concentration of 2 μg / mL in a 96-well plate. For each sample, make two replicates and one blank background well without antigen stimulants. Stimulate the positive control wells with 1 μL of PMA / Ionomycin Mixture (Lianke Biotechnology: CS1001). Slowly drip 200 μL of the mixed antigen stimulant spleen cell suspension into the wells using a pipette along the side wall of the Elispot plate. Then, place the plate in a cell culture incubator at 37°C and 5% CO2 for 36 hours. During this process, ensure aseptic conditions and do not move the plate. Following the kit instructions, the plates were washed and incubated with antibodies. After staining and air-drying in the dark, cell counting was performed using a fluorescent enzyme-linked immunosorbent assay (ELISA) analyzer. The number of spots in wells with added stimulants was subtracted from the number of spots in wells without stimulants, and the results were summarized and analyzed to calculate the number of T cells specifically secreting IFN-γ and IL-4 per million spleen cells. Results are as follows: Figure 4 As shown, compared with the CpG / Alum negative control group, the gB2, gD2, and gE2 experimental groups of the present invention induced higher levels of IFN-γ and IL-4 responses, which were comparable to those of the gC2, gD2, and gE2 positive control groups.

[0082] 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. An HSV-2 triantigen subunit vaccine composition, characterized in that, The vaccine comprises HSV-2 recombinant protein and a complex adjuvant; the HSV-2 recombinant protein consists of the extracellular domains of three proteins: HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2, wherein the amino acid sequences of the extracellular domains of HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the complex adjuvant consists of CpG oligodeoxynucleotides and aluminum adjuvant. The mass ratio of HSV-2 gB2 protein extracellular domain, HSV-2 gD2 protein extracellular domain, HSV-2 gE2 protein extracellular domain, CpG oligodeoxynucleotide, and aluminum adjuvant is 5:5:5:50:

125.

2. The method for preparing the HSV-2 triantigen subunit vaccine composition according to claim 1, characterized in that, Includes the following steps: Step (1) The coding gene sequences corresponding to the extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 were cloned into the HindIII and EcoRI restriction sites of the pFastBac1 plasmid to construct recombinant pFastBac1gB2 plasmid, recombinant pFastBac1gD2 plasmid, and recombinant pFastBac1gE2 plasmid; Step (2): Obtain the target fragments of the three protein-coding genes from the recombinant pFastBac1gB2 plasmid, the recombinant pFastBac1gD2 plasmid, and the recombinant pFastBac1gE2 plasmid, respectively, and insert them into the Bacmid plasmid to construct the three recombinant Bacmid plasmids. Step (3): The three recombinant Bacmid plasmids constructed in step (2) were expressed by the eukaryotic expression system sf9 cells. The proteins were purified by nickel column purification to obtain the extracellular domains of HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 respectively. Step (4): The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 obtained in step (3) are dissolved in phosphate buffer and a compound adjuvant is added to it before immunization to prepare HSV-2 triantigen subunit vaccine.

3. The method for preparing the HSV-2 triantigen subunit vaccine composition according to claim 2, characterized in that, Dissolve 5 μg each of the extracellular domains of the three HSV-2 proteins gB2, gD2, and gE2 obtained in step (3) in phosphate buffer to prepare a 15 μg / injection dose; before immunization, add 50 μg / injection of compound adjuvant CpG oligodeoxynucleotide and 125 μg / injection of aluminum adjuvant to prepare the HSV-2 triantigen subunit vaccine.

4. The use of the HSV-2 triantigen subunit vaccine composition of claim 1 in the preparation of a vaccine for the prevention or treatment of diseases caused by herpes simplex virus infection.

5. The use of the HSV-2 triantigen subunit vaccine composition according to claim 1 in the preparation of a vaccine for the prevention or treatment of diseases caused by herpes simplex virus infection, characterized in that, Herpes simplex virus includes herpes simplex virus type 1 and herpes simplex virus type 2.

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