HSV-2 trivalent mucosal vaccine composition based on flic / cpg adjuvant system and uses thereof

Abstract

The present application relates to HSV-2 trivalent mucosal vaccine composition based on FliC / CpG adjuvant system and application thereof, and belongs to the technical field of biological medicine. The vaccine comprises Salmonella typhimurium flagellin FliC, CpG oligonucleotide and HSV-2 gC2-gD2-gE2 triple antigen tandem recombinant protein; wherein the amino acid sequence of Salmonella typhimurium flagellin FliC is shown as SEQ ID NO. 1; the sequence of CpG oligonucleotide is shown as SEQ ID NO. 3. The HSV-2 trivalent mucosal vaccine based on FliC / CpG adjuvant system can effectively induce mucosal immunity of mice and has significant protection efficacy against HSV-2 / KM-1 challenge after intranasal immunization of BALB / c mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a trivalent HSV-2 mucosal vaccine composition based on the FliC / CpG adjuvant system and its application. Background Technology

[0002] Herpes simplex virus type 2 (HSV-2) belongs to the genus Herpes simplex virus in the subfamily Alphaherpesvirinae of the family Herpesviridae. It is an enveloped double-stranded DNA virus with a genome length of approximately 152 kb. HSV-2 is the main cause of genital herpes, and its infection rate is increasing year by year. Harfouche et al. reported in 2025 that the total number of infections in 2020 accounted for 13.3% of the world's population.

[0003] Vaccines are the most economical and effective means of preventing genital herpes. Given the widespread prevalence of the genital herpes virus and its difficulty in complete eradication due to its latent infection characteristics, vaccines are the optimal solution. Currently, the four most advanced preventative vaccines—the subunit vaccine GSK208141, the gB2 / gD2 subunit vaccine, and the mRNA vaccine GSK-4108771A—have all terminated their clinical trials. This means that the development of a genital herpes vaccine is both challenging and extremely urgent.

[0004] The Friedman lab at the University of Pennsylvania proposed a novel vaccine design concept—no longer focusing solely on blocking viral entry, but simultaneously blocking the virus's two core immune escape mechanisms, constructing a "three-in-one" multi-target defense system: inducing gC2 antibodies to overcome complement escape, gD2 antibodies to prevent viral entry, and gE2 antibodies to restore antibody-dependent cytotoxicity. Their results showed that the gC2, gD2, and gE2 combination antigen exhibited excellent protective efficacy. However, the gC2, gD2, and gE2 protein combination antigen suffers from high production costs and complex production lines. Furthermore, their attempt did not consider the importance of inducing mucosal immunity for HSV-2 vaccine design, meaning that this gC2, gD2, and gE2 combination antigen remains far from becoming a qualified HSV-2 vaccine candidate.

[0005] Therefore, overcoming the shortcomings of existing technologies is an urgent problem to be solved in the field of biomedicine. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an HSV-2 trivalent mucosal vaccine composition based on the FliC / CpG adjuvant system and its application.

[0007] 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 trivalent mucosal vaccine composition for HSV-2 based on the FliC / CpG adjuvant system, comprising Salmonella typhimurium flagellate protein FliC, CpG oligonucleotides and HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein; The amino acid sequence of the flagellated protein FliC of Salmonella Typhimurium is shown in SEQ ID NO.1; The sequence of the CpG oligonucleotide is shown in SEQ ID NO.3.

[0008] Furthermore, the mass ratio of Salmonella Typhimurium flagellate protein FliC, CpG oligonucleotide, and HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein was 1:10:15.

[0009] The second aspect of the present invention provides a method for preparing a trivalent HSV-2 mucosal vaccine based on a FliC / CpG adjuvant system, using the aforementioned trivalent HSV-2 mucosal vaccine composition based on a FliC / CpG adjuvant system, wherein the trivalent HSV-2 mucosal vaccine based on a FliC / CpG adjuvant system includes an nasal drop vaccine and a subcutaneous vaccine; The specific preparation method is as follows: HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, Salmonella typhimurium flagellate protein FliC, and CpG oligonucleotides were dissolved in physiological saline until the ratio of total volume to CpG oligonucleotide mass was 70 μL: 10 μg to obtain the nasal drop vaccine. HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, Salmonella typhimurium flagellate protein FliC, and CpG oligonucleotides were dissolved in physiological saline until the ratio of total volume to CpG oligonucleotide mass was 100 μL: 10 μg, thus obtaining the subcutaneous vaccine.

[0010] Further, 15 μg of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 1 μg of Salmonella typhimurium flagellate protein FliC, and 10 μg of CpG oligonucleotide were dissolved in physiological saline to a total volume of 70 μL to obtain the nasal drop vaccine; each dose of nasal drop vaccine has a volume of 70 μL. Dissolve 15 μg HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 1 μg Salmonella typhimurium flagellate protein FliC, and 10 μg CpG oligonucleotide in physiological saline to a total volume of 100 μL to obtain a subcutaneous vaccine; each dose of subcutaneous vaccine has a volume of 100 μL.

[0011] The third aspect of the present invention provides a method for preparing the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system, and the resulting HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system.

[0012] The fourth aspect of the present invention provides the use of the HSV-2 trivalent mucosal vaccine composition based on the FliC / CpG adjuvant system in the preparation of a vaccine for the prevention or treatment of diseases caused by infection with herpes simplex virus type 2.

[0013] This invention provides a trivalent HSV-2 mucosal vaccine composition based on the FliC / CpG adjuvant system and its application. This invention fuses and expresses gC2, gD2, and gE2 proteins to construct an HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein as the vaccine antigen. Regarding the adjuvant selection, the novel adjuvant Salmonella typhimurium flagellate protein (FliC) is chosen primarily for Th2-type immune activation, while the classic adjuvant CpG oligonucleotides are used for Th1-type immune complementation. This combination represents a completely new approach in the field of HSV-2 vaccine research.

[0014] Unlike monoantigen vaccines, the vaccine composition of this invention induces gC2 antibody to relieve complement escape, gD2 antibody to prevent viral entry, and gE2 antibody to restore antibody-dependent cytotoxicity (ADCC), thus constructing a multi-target defense system in a "three-in-one" manner.

[0015] The HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein of this invention is prepared according to the method described in the following literature: Shi Jiandong et al. An HSV-2 triantigen tandem recombinant protein vaccine composition and its application: ZL 2026 1 0277038.6 [P]. 2026-06-12.

[0016] This invention provides two immunization methods for the above-mentioned HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system in the BALB / c mouse model: intranasal immunization and subcutaneous immunization.

[0017] This invention applies the clinical isolate HSV-2 / KM-1 to the protective efficacy evaluation of this HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system. In selecting challenge strains, traditional laboratory reference strains are often used as a relatively stable and homogeneous "gold standard," whose core function is to verify the universal protective efficacy of the vaccine. However, after years of passage and adaptation in cells, their virulence characteristics may differ from the latest prevalent clinical strains. Therefore, this invention incorporates the clinical isolate HSV-2 / KM-1 into the vaccine protective efficacy evaluation to reflect the vaccine's protective efficacy against the latest prevalent strains.

[0018] Compared with the prior art, the beneficial effects of this invention are as follows: 1. The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system of this invention significantly increased the level of secretory IgA (sIgA) in vaginal irrigation fluid after immunizing mice via nasal drop, while the subcutaneous immunization group showed a weak induction effect on sIgA in the vagina. This indicates that the present invention can effectively activate the common mucosal immune system through nasal drop immunization and establish the first line of immune defense at the initial site of HSV-2 infection—the reproductive tract mucosa.

[0019] 2. The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system of this invention can significantly upregulate the secretion levels of Th1 cytokines IFN-γ and IL-2 and Th2 cytokines IL-10 in spleen cells when administered via nasal drop immunization; while the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system can only upregulate the secretion of IL-2 and IL-10 by spleen cells when administered via subcutaneous immunization, and has limited ability to induce the key antiviral factor IFN-γ.

[0020] 3. Following intranasal or subcutaneous immunization of mice with the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system of this invention, the serum titers of specific IgG, IgG1, and IgG2a antibodies significantly increased 42 days after the initial immunization, exhibiting a characteristic of IgG1 > IgG2a, indicating that this invention can induce a humoral immune response predominantly Th2-type. Furthermore, the serum IgA level in the intranasal immunization group was significantly higher than that in the subcutaneous immunization group, further confirming that the intranasal immunization route can more effectively stimulate a systemic mucosal immune response.

[0021] 4. In this invention, mice immunized with the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system via intranasal and subcutaneous administration were subjected to a 15-fold median lethal dose (15×LD50). 50 The HSV-2 / KM-1 clinical isolates were challenged vaginally. In the nasal drop vaccine group, all 10 mice survived 14 days after challenge with the antigen-adjuvant combination. In the subcutaneous vaccine group, 9 out of 10 mice survived 14 days after challenge with the antigen-adjuvant combination. One mouse died. A single-variable control group was also established, consisting of adjuvants used in the same dosage and immunization method as the nasal drop vaccine group (or subcutaneous vaccine group), except for the absence of antigen. All mice in the nasal drop and subcutaneous adjuvant groups died on day 12 after challenge. These results demonstrate that the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system provided by this invention can provide highly effective protection for mice and significantly reduce the mortality rate caused by HSV-2 infection.

[0022] 5. The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system of this invention can effectively inhibit viral replication and accelerate viral clearance. Post-challenge vaginal viral shedding monitoring results showed that the peak viral load in the vagina of mice in the nasal drop vaccine group and the subcutaneous vaccine group occurred on day 2 after challenge, then rapidly decreased from day 4 to day 8, and was completely cleared by day 10; while the peak viral shedding in the vagina of mice in the nasal drop adjuvant group and the subcutaneous adjuvant group occurred on day 4, after which a high viral nucleic acid copy number was maintained. These results indicate that this invention can effectively control HSV-2 replication in vivo and accelerate viral clearance.

[0023] 6. The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system of this invention can significantly delay the occurrence and progression of genital lesions. The onset of genital lesions (days 7-8) in mice in the nasal and subcutaneous vaccine groups was significantly later than that in the nasal and subcutaneous adjuvant groups (day 3), and the severity of the lesions was significantly reduced. These results indicate that this invention can effectively delay genital lesions caused by HSV-2 infection and reduce the severity of the disease.

[0024] 7. After three immunizations with the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system described in this invention via intranasal or subcutaneous administration, no obvious pathological damage was observed in the major organs of mice, including the heart, liver, spleen, lungs, and kidneys. Attached Figure Description

[0025] Figure 1 Figure A shows the results of detecting secretory sIgA and sIgG antibodies in vaginal lavage fluid; Figure B shows the comparison of OD values ​​of secretory sIgA in vaginal lavage fluid detected by ELISA in the nasal drop vaccine group, nasal drop adjuvant group, nasal drop saline group, subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of this invention; Figure C shows the comparison of OD values ​​of secretory sIgG in vaginal lavage fluid detected by ELISA in the nasal drop vaccine group, nasal drop adjuvant group, nasal drop saline group, subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of this invention.

[0026] Figure 2Figure A shows the serum titers of bound antibody IgA and IgG in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group at days 14, 28, and 42 after the first immunization; Figure B shows the serum titers of bound antibody IgA in the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group at days 14, 28, and 42 after the first immunization; Figure C shows the serum titers of bound antibody IgA in the nasal drop vaccine group and subcutaneous vaccine group at days 14, 28, and 42 after the first immunization. Figure D shows the results of the IgA titer of the conjugated antibody; Figure E shows the results of the IgG titer of the conjugated antibody in the serum of the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group on days 14, 28, and 42 after the first immunization; Figure F shows the results of the IgG titer of the conjugated antibody in the serum of the nasal drop vaccine group and subcutaneous vaccine group on days 14, 28, and 42 after the first immunization. Figure 3 Figure A shows the serum titer results of binding antibodies IgG1 and IgG2a on day 42 after the first immunization in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group of the present invention; Figure B shows the serum titer results of binding antibodies IgG1 in the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of the present invention on day 42 after the first immunization; Figure C shows the serum titer results of binding antibodies IgG2a in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group of the present invention on day 42 after the first immunization; Figure D shows the serum titer results of binding antibodies IgG2a in the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of the present invention on day 42 after the first immunization; Figure E shows the serum titer results of binding antibodies IgG1 and IgG2a in the nasal drop vaccine group and subcutaneous vaccine group of the present invention on day 42 after the first immunization. Figure 4 Figure A shows the serum neutralizing antibody titer results for the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group on day 42 after the first immunization; Figure B shows the serum neutralizing antibody titer results for the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group on day 42 after the first immunization; Figure C shows a comparison of the serum neutralizing antibody titer results for the nasal drop vaccine group and subcutaneous vaccine group on day 42 after the first immunization. Figure 5Figure A shows the results of spleen cell stimulation supernatant cytokine secretion level detection; Figure B shows the results of spleen cell stimulation supernatant IFN-γ secretion level in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group of the present invention on day 42 after the first immunization; Figure B shows the results of spleen cell stimulation supernatant IFN-γ secretion level in the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of the present invention on day 42 after the first immunization. C shows the IFN-γ secretion level in the supernatant stimulated by spleen cells on day 42 after the first immunization in the nasal drop vaccine group and subcutaneous vaccine group of the present invention; D shows the IL-2 secretion level in the supernatant stimulated by spleen cells on day 42 after the first immunization in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group of the present invention; E shows the IL-2 secretion level in the supernatant stimulated by spleen cells on day 42 after the first immunization in the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of the present invention; F shows the IL-2 secretion level in the supernatant stimulated by spleen cells on day 42 after the first immunization in the nasal drop vaccine group and subcutaneous vaccine group of the present invention; G shows the IL-4 secretion level in the supernatant stimulated by spleen cells on day 42 after the first immunization in the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group of the present invention. H represents the IL-4 secretion level in the spleen cell supernatant on day 42 after the first immunization of the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of this invention; I represents the IL-4 secretion level in the spleen cell supernatant on day 42 after the first immunization of the nasal vaccine group and subcutaneous vaccine group of this invention; J represents the IL-10 secretion level in the spleen cell supernatant on day 42 after the first immunization of the nasal vaccine group, nasal adjuvant group, and nasal saline group of this invention; K represents the IL-10 secretion level in the spleen cell supernatant on day 42 after the first immunization of the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of this invention; L represents the IL-10 secretion level in the spleen cell supernatant on day 42 after the first immunization of the nasal vaccine group and subcutaneous vaccine group of this invention. Figure 6 The diagram shows the protective evaluation results of the nasal drop vaccine group, nasal drop adjuvant group, subcutaneous vaccine group, and subcutaneous adjuvant group of this invention. In the diagram, A represents the change in mouse body weight within 14 days after HSV-2 / KM-1 virus challenge; B represents the change in mouse reproductive tract lesion score within 14 days after HSV-2 / KM-1 virus challenge; C represents the change in viral load in vaginal swabs within 14 days after HSV-2 / KM-1 virus challenge; D represents the viral load in the dorsal root ganglia of mice on day 7 after HSV-2 / KM-1 virus challenge; E represents the viral load in the vagina of mice on day 7 after HSV-2 / KM-1 virus challenge; F represents the viral load in the uterus of mice on day 7 after HSV-2 / KM-1 virus challenge; and G represents the viral load in the bladder of mice on day 7 after HSV-2 / KM-1 virus challenge. Figure 7These are pathological sections of the heart, liver, spleen, lung, and kidney after the completion of the immunization procedures for the nasal drop vaccine group, nasal drop adjuvant group, nasal drop saline group, subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group of the present invention. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the embodiments.

[0028] 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.

[0029] The main cells and reagents used in the embodiments of this invention are as follows: Vero cells (African green monkey kidney cells) are cultured using Durbecco's modified eagle medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution (PS).

[0030] Antibodies: HRP-labeled IgG, IgA, IgG1, and IgG2a antibodies were purchased from Abcam Antibody Company, with catalog numbers ab6789, ab97235, ab97240, and ab97245, respectively.

[0031] PBS (containing phenol red), PS, and 0.25% (v / v) trypsin were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences; DMEM (catalog number: C3113-0500) and fetal bovine serum (catalog number: C04001-050) were purchased from Shanghai Xiaopeng Biotechnology Co., Ltd. Nucleic acid extraction or purification kit (catalog number: M2006P-96KF) was purchased from Xinbaiji Biotechnology Co., Ltd. ELISA stop solution (catalog number: C1058), ELISA coating solution (C1055), and TMB chromogenic solution (catalog number: PR1210) were purchased from Beijing Solarbio Science & Technology Co., Ltd. The mouse Th1 / Th2 uncoated kit (catalog number: 88-7711-44) was purchased from Thermo Fisher Scientific China Co., Ltd.

[0032] Example 1: Preparation of a trivalent HSV-2 mucosal vaccine based on the FliC / CpG adjuvant system 1. Preparation of HSV-2 gC2-gD2-gE2 tandem recombinant protein expression The HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein of this invention is prepared according to the method described in the following literature: Shi Jiandong et al. An HSV-2 triantigen tandem recombinant protein vaccine composition and its application: ZL 2026 1 0277038.6 [P]. 2026-06-12; 2. Preparation of FliC expression from Salmonella Typhimurium The FliC in this invention was expressed and purified to a purity greater than 85% and endotoxin was removed by Suzhou Hongxun Biotechnology Co., Ltd. using a prokaryotic expression system.

[0033] The molecular weight of the Salmonella typhimurium flagellin FliC is 55.3 kDa, and its full-length amino acid sequence (including the tag sequence) is as follows: (SEQ ID NO.1).

[0034] The full-length nucleotide sequence of FliC expression (including the tag sequence) is as follows:

[0035] 3. CpG Oligonucleotide Acquisition CpG oligonucleotides were purchased from Suzhou Hongxun Biotechnology Co., Ltd., and their sequences are as follows: 5'-tccatgacgttcctgacgtt-3' (SEQ ID NO.3) 4. Vaccine preparation plan The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system was prepared by dissolving 15 μg of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 1 μg of Salmonella typhimurium flagellin FliC, and 10 μg of CpG oligonucleotides in physiological saline. The volume of physiological saline used is detailed in the immunization protocol of Example 2.

[0036] Example 2: Evaluation of vaccine immunogenicity 1. Immunization regimen Thirty-six 6-8 week old BALB / c female mice weighing 18-20g were randomly divided into 6 groups of 6 mice each. Based on the immunization method, they were further divided into intranasal and subcutaneous administration. Based on the immunization content, they were further divided into vaccine, adjuvant, and saline administration. Therefore, the 6 groups were: intranasal vaccine group, intranasal adjuvant group, intranasal saline group, subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group. Due to the different immunization methods, there were practical difficulties in implementation, as intranasal immunization required a small injection volume while subcutaneous immunization required an injection volume that was a multiple of 50μL. Ultimately, the injection volume used for intranasal immunization was 70μL, and the injection volume used for subcutaneous immunization was 100μL. The extra 30μL for subcutaneous immunization was supplemented by the solvent (saline). Immunization was administered on days 0, 14, and 28, for a total of three times.

[0037] The immunization details for each group are as follows: Table 1 Immunization Content

[0038] 1. Detection of mucosal immune antibody levels in vaginal douche fluid The animals were sacrificed 14 days after the last immunization (i.e., 42 days after the first immunization), and the vagina was irrigated with 200 μL PBS. The vaginal irrigation fluid was collected and used as the test vaginal irrigation fluid.

[0039] (1) Coat the ELISA plate wells with HSV-2 gC2-gD2-gE2 triple antigen tandem recombinant protein (100ng / well) and incubate overnight at 4°C.

[0040] (2) The next day, after washing the plate 5 times with PBST, it was sealed with 2% (w / v) BSA at 200 μL / well at 37°C for 2 hours.

[0041] (3) Wash the plate 3 times with PBST, add 100 μL of vaginal irrigation solution per well (for sIgA) or 50 μL per well (for sIgG) to the coated well, and incubate at 37°C for 1 hour.

[0042] (4) After washing the plate 5 times with PBST, incubate to detect the antibody. Dilute the HRP-labeled goat anti-mouse secondary antibody with 2% (w / v) BSA: for HRP-labeled goat anti-mouse IgG secondary antibody, the volume ratio of HRP-labeled goat anti-mouse IgG secondary antibody to 2% (w / v) BSA is 1:100000; for HRP-labeled goat anti-mouse IgA secondary antibody, the volume ratio of HRP-labeled goat anti-mouse IgA secondary antibody to 2% (w / v) BSA is 1:50000. The final diluted volume of HRP-labeled goat anti-mouse secondary antibody is 100 μL / well, and incubate at 37°C for 1 hour.

[0043] (5) Wash the plate 5 times with PBST, pat dry, add 100 μL of TMB colorimetric solution to each well, react in the dark for 4-5 minutes, add 50 μL of ELISA stop solution to each well, measure the OD value at 450 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, and compare the OD values ​​of each group.

[0044] The results are as follows Figure 1 As shown; Figure 1 In the study, the nasal drop vaccine group showed significant IgA secretion, establishing a defense against HSV-2 infection, while the subcutaneous vaccine group showed only moderate results, differing only from the adjuvant group and not statistically significantly from the saline group. Compared to the subcutaneous vaccine group, the nasal drop vaccine induced significantly higher levels of sIgA. Figure 1 In the B group, both the intranasal and subcutaneous immunization groups induced significant sIgG, but there was no significant difference in sIgG levels between the two groups. Therefore, overall, the intranasal immunization method is more likely to induce mucosal secretory antibodies.

[0045] 2. Serum binding antibody level detection Mice serum was collected at three time points: day 14, day 28, and day 42 after the first immunization.

[0046] (1) Coat the ELISA plate wells with HSV-2 gC2-gD2-gE2 triple antigen tandem recombinant protein (100ng / well) and incubate overnight at 4°C.

[0047] (2) The next day, after washing the plate 5 times with PBST, it was sealed with 2% (w / v) BSA at 200 μL / well at 37°C for 2 hours.

[0048] (3) Wash the plate 3 times with PBST. Dilute the serum to be tested 2 times starting from 1:20 (for IgA), 1:2000 (for IgG), or 1:8000 (for IgG1 or IgG2a) to 1:2560 (for IgA), 1:256000 (for IgG), or 1:1024000 (for IgG1 and IgG2a). Inoculate the serum of the 8 dilutions into each well of a row of 96-well plates, i.e., one well for each dilution. Incubate at 37°C for 1 hour.

[0049] (4) After washing the plate 5 times with PBST, incubate to detect antibodies: dilute HRP-labeled goat anti-mouse secondary antibody (IgG 1:100000; IgA, IgG1, and IgG2a 1:50000) with 2% (w / v) BSA, 100 μL / well, and incubate at 37°C for 1 hour.

[0050] (5) Wash the plate 5 times with PBST, pat dry, add 100 μL of TMB chromogenic solution to each well, react in the dark for 4-5 minutes, add 50 μL of ELISA stop solution to each well, and measure the OD value at 450 nm using a microplate reader. The OD value of the positive well is greater than 2.1 times that of the negative well.

[0051] like Figure 2 A in Figure 2 B in Figure 2 As shown in C, subcutaneous immunization with subcutaneous vaccine, subcutaneous adjuvant, and subcutaneous saline failed to induce serum IgA, indicating that systemic mucosal immunity was not initiated. In contrast, the serum IgA level in the nasal drop vaccine group increased significantly with increasing immunization frequency and duration. Figure 2 D in Figure 2 E in Figure 2 As shown in F, both the nasal drop and subcutaneous vaccine groups showed a significant increasing trend in serum IgG binding antibody titers with increasing immunization frequency and time, compared to the nasal drop adjuvant group, nasal drop saline group, subcutaneous adjuvant group, and subcutaneous saline group. There was no significant difference in serum IgG antibody levels between the nasal drop and subcutaneous vaccine groups on day 42 after the first immunization. Figure 3 A in Figure 3 B in Figure 3 C in Figure 3 The results in D showed that both the nasal drop vaccine group and the subcutaneous vaccine group induced significantly increased serum IgG1 and IgG2a binding antibody titers compared to the nasal drop adjuvant group, the nasal drop saline group, the subcutaneous adjuvant group, and the subcutaneous saline group, respectively. Figure 3As shown in E, both the intranasal vaccine group and the subcutaneous vaccine group showed a slight trend of IgG1 levels being higher than IgG2a levels in serum, but this was not statistically significant. Therefore, from the perspective of systemic immune response, there was no significant difference in the induction levels of serum-specific IgG, IgG1, and IgG2a between the two immunization methods.

[0052] 3. Neutralization experiment Neutralizing antibody tests were performed on serum from six groups of mice collected 42 days after the first immunization: the nasal vaccine group, the nasal adjuvant group, the nasal saline group, the subcutaneous vaccine group, the subcutaneous adjuvant group, and the subcutaneous saline group. The serum collected at this time was recorded as the test serum.

[0053] (1) The serum to be tested was inactivated at 56℃ for 30 min to remove non-specific inhibitors.

[0054] (2) In a 96-well plate, starting from the first row, use DMEM medium to start with a 1:5 dilution, and then perform 2-fold serial dilutions to 1:640. Each tube of serum to be tested has 4 replicates.

[0055] (3) Then add 50 μL of virus to each well, for a total of 100 TCID. 50 HSV-2 / KM-1 viral fluid (concentration of 2000 TCID) 50 ( / mL), incubate at 37℃ for 1 hour.

[0056] (4) Virus back-drop: Dilute to a concentration of 100 TCID 50 50 μL of HSV-2 / KM-1 virus solution was serially diluted 10-fold (10 TCID50). 50 / 50μL, 1TCID 50 / 50μL, 0.1TCID 50 Add 50 μL of the solution to a 96-well plate, bring the volume to 100 μL with DMEM, and incubate at 37°C for 1 hour.

[0057] (5) Add 100 μL of a solution with a concentration of 4 × 10⁻⁶ to the well. 4 Cells were cultured in 100 μL of DMEM medium per cell culture medium at 37°C for 4 days, and cell pathogenesis was observed.

[0058] (6) Result interpretation: The highest serum dilution that can protect 50% of cells from lesions is the neutralization endpoint. The result is calculated according to the Reed-Muench method.

[0059] (7) Reed-Muench calculation method: Calculate the cumulative number of diseased wells (a) for each dilution from low dilution (1:5) to high dilution (1:640), and the cumulative number of non-diseased wells (b) for each dilution from high dilution to low dilution. Convert the a:b ratio for each dilution to a percentage, which is the disease rate (p) for that dilution. The maximum disease rate less than 50% is denoted as p1, and the minimum disease rate greater than 50% is denoted as p2. Then, calculate the distance ratio (d) as (50% - p1) / (p2 - p1). Let L be the logarithm of the dilution with a disease rate less than 50%, and K be the logarithm of the dilution factor. The formula for calculating the neutralizing antibody titer is: lg(50% serum neutralization endpoint) = L + d × K The results are as follows Figure 4 As shown. Figure 4 Figure A in the figure shows the serum neutralizing antibody titers of the nasal drop vaccine group, nasal drop adjuvant group, and nasal drop saline group on day 42 after the first immunization. Figure 4 B in the figure represents the serum neutralizing antibody titer results of the subcutaneous vaccine group, subcutaneous adjuvant group, and subcutaneous saline group on day 42 after the first immunization. Figure 4 C in the figure represents a comparison of serum neutralizing antibody titers in the nasal drop vaccine group and the subcutaneous vaccine group on day 42 after the first immunization. Both nasal drop and subcutaneous immunization methods can induce the production of serum neutralizing antibodies. The highest serum neutralizing antibody titers induced by the nasal drop vaccine and the subcutaneous vaccine are only 1:50 and 1:28, respectively. Overall, there is no significant difference in serum neutralizing antibody levels between the nasal drop vaccine and the subcutaneous vaccine.

[0060] 4. Detection of cytokine levels in spleen cell-stimulated supernatant Mice for immunogenicity analysis were sacrificed 14 days after their last immunization, and spleen cells were isolated and administered HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein (3 μg / 1×10⁻⁶). 7Stimulation with spleen cells for 48 hours. Following the Thermo Fisher Scientific mouse Th1 / Th2 cytokine uncoated kit instructions, 100 μL of the corresponding cytokine capture antibody was coated onto each well of an ELISA plate at 4°C the night before. 200 μL of washing buffer was added to each well of the ELISA plate and discarded; this was the first wash. After washing three times, the plates were blocked with 200 μL of ELISA diluent per well at room temperature for 1 hour. The standard stock solutions in the kit (concentrations of the cytokine standard stock solutions were IFN-γ: 2000 pg / mL; IL-2: 200 pg / mL; IL-4: 500 pg / mL; IL-10: 4000 pg / mL) were serially diluted 2-fold to 64-fold, resulting in six dilutions of the standard stock solutions: 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold. Subsequently, 100 μL of standard stock solution, serially diluted standard stock solutions (6 dilutions), and test samples (the test samples were 1×10⁻⁶ of each mouse stimulated for 48 hours with HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein) were added. 7 Add 100 μL of cell culture supernatant (from one spleen cell) to each well and incubate at room temperature for 2 hours. Wash the plate four times with washing buffer, then add 100 μL of detection antibody to each well, seal the plate, and incubate at room temperature for 1 hour. Wash the plate three times, soaking for 1 minute each time. Add 100 μL of streptavidin solution from the kit to each well, seal the plate, and incubate at room temperature for 30 minutes. Wash the plate five times, soaking for 1 minute each time. Add 100 μL of TMB substrate solution, seal the plate, and incubate at room temperature for 15 minutes. Finally, add 100 μL of stop solution. Read and calculate immediately at 450 nm.

[0061] The results are as follows Figure 5 As shown, the levels of IFN-γ and IL-2 induced by nasal immunization were significantly higher than those induced by subcutaneous immunization, and subcutaneous immunization failed to induce IFN-γ. Figure 5 A- Figure 5 (F in the original text) The nasal drop immunization method has a higher ability to induce Th1-type immunity than the subcutaneous immunization method. For Th2-type immunity, the nasal drop immunization group has a significantly better ability to upregulate IL-10 secretion levels than the subcutaneous immunization group. However, in terms of IL-4 stimulation, neither the nasal drop immunization group nor the subcutaneous immunization group induced IL-4 secretion levels significantly higher than those induced by the nasal drop adjuvant group, nasal drop saline group, subcutaneous adjuvant group, or subcutaneous saline group. Therefore, the nasal drop immunization group has a greater advantage than the subcutaneous immunization group in inducing Th2-type immune responses.

[0062] Example 3: Evaluation of vaccine protective efficacy 1. Immunization regimen Evaluation of protection against viral challenge after 14 days: Forty 6-8 week old BALB / c female mice were randomly divided into four groups of 10 each. Based on the immunization method, they were further divided into intranasal and subcutaneous administration. Based on the immunization content, they were further divided into vaccine and adjuvant administration. Therefore, the four groups were: intranasal vaccine group, intranasal adjuvant group, subcutaneous vaccine group, and subcutaneous adjuvant group. Immunization was administered on days 0, 14, and 28, for a total of three administrations.

[0063] 7-day challenge protection evaluation: Forty 6-8 week old BALB / c female mice were randomly divided into four groups of 10 mice each. Based on the immunization method, they were further divided into intranasal and subcutaneous administration. Based on the immunization content, they were further divided into vaccine and adjuvant administration. Therefore, the four groups were: intranasal vaccine group, intranasal adjuvant group, subcutaneous vaccine group, and subcutaneous adjuvant group. Immunization was administered on days 0, 14, and 28, for a total of three times. Two mice died unexpectedly during the immunization phase, leaving 10 mice in the intranasal vaccine group, 9 mice in the intranasal adjuvant group, 10 mice in the subcutaneous vaccine group, and 9 mice in the subcutaneous adjuvant group for the final 7-day challenge protection evaluation.

[0064] The immunization details for each group are as follows: Table 2 Immunization Content

[0065] 2. Virus attack plan Evaluation of protection against 14-day challenge: Mice were vaginally challenged 14 days after the last immunization (i.e., 42 days after the first immunization) with a challenge dose of 15 times the LD50 (7.05 × 10⁻⁶). 3 PFU / mouse). The body weight of mice in each group was recorded daily for 14 days after challenge, and lesion scores were calculated (see Table 3 for scoring criteria). Vaginal swabs were collected on days 2, 4, 6, 8, 10, 12, and 14 after challenge to detect virus shedding.

[0066] Evaluation of protection against 7-day challenge: Mice were vaginally challenged 14 days after the last immunization (i.e., 42 days after the first immunization) at a challenge dose of 15 times the LD50 (7.05 × 10⁻⁶). 3 PFU / mouse). For 7 consecutive days after challenge, the body weight of mice in each group was recorded and lesion scores were calculated (scoring criteria are shown in Table 3). Mice were sacrificed on day 7 after challenge, and tissue samples from the vagina, uterus, bladder, and dorsal root ganglion were collected for viral load determination.

[0067] Table 3 Scoring Criteria for Genital Lesions

[0068] The results are as follows Figure 6 As shown. Figure 6In the A group, monitoring of mouse weight and genital lesions after challenge revealed that mice in both the nasal vaccine and adjuvant groups began to lose weight from day 3 onwards, with the adjuvant group showing a faster rate of weight loss. In the subcutaneous vaccine group, weight loss began from day 4 post-challenge, with the subcutaneous adjuvant group showing weight loss from day 3 onwards and at a faster rate than the subcutaneous vaccine group. Compared to the subcutaneous vaccine group, although the nasal vaccine group experienced weight loss earlier, the rate of weight loss was slower.

[0069] like Figure 6 In section B, based on genital lesion scores, both adjuvant groups showed lesion characteristics starting from day 3, with rapid progression leading to death. The nasal drop vaccine group showed mild lesion characteristics starting from day 8, while the subcutaneous vaccine group showed lesion characteristics starting from day 7. The peak lesion scores for both groups were similar, with the nasal drop vaccine group showing a slightly higher peak score. However, since the observation period ended at 14 days, subsequent disease progression was unknown. Based on the current lesion characteristics, the two immunization methods showed little difference in their effectiveness in delaying lesions.

[0070] like Figure 6 In mice receiving either the nasal or subcutaneous adjuvant (C), the peak viral shedding occurred on day 4. Viral copy number subsequently decreased but did not reach zero, remaining at a high level. In contrast, the peak viral copy number for the nasal and subcutaneous vaccines occurred on day 2. Vaginal viral shedding improved subsequently, with a rapid decline in viral copy number between days 4 and 8. By day 10, vaginal viral shedding in both vaccine groups had reached zero. Therefore, the vaccines are effective in controlling viral replication in mice.

[0071] like Figure 6 D~ Figure 6 In the study of G, regarding nerve tissue, the viral load in the dorsal root ganglia of the nasal drop vaccine group was significantly lower than that of the nasal drop adjuvant group and the subcutaneous vaccine group. The viral load in the dorsal root ganglia of the subcutaneous vaccine group was also significantly lower than that of the subcutaneous adjuvant group. This suggests that nasal drop immunization is more effective than subcutaneous immunization in preventing acute viral invasion of the nervous system. Viral load results in the bladder, vagina, and uterus showed significantly higher copies compared to the nasal drop and subcutaneous vaccine groups, respectively. There was no significant difference in viral load between the subcutaneous and nasal drop vaccine groups in these three tissues. Therefore, this invention proposes that while there is no significant difference between nasal drop and subcutaneous immunization in protecting the reproductive and urinary systems, nasal drop immunization has a significant advantage in preventing acute viral invasion of the nervous system.

[0072] Example 4: Vaccine Safety Analysis The immunization grouping and immunization content in this embodiment are exactly the same as those in Example 2 (Table 1). The animals were sacrificed 42 days after the first immunization, and their hearts, livers, spleens, lungs, and kidneys were collected for HE staining to analyze the pathological damage of each organ.

[0073] The results are as follows Figure 7 As shown, there were no significant differences in the pathological findings of the heart, liver, spleen, lungs, and kidneys among the nasal vaccine group, nasal adjuvant group, subcutaneous vaccine group, subcutaneous adjuvant group, nasal saline group, and subcutaneous saline group. This indicates that neither the vaccine, adjuvant, nor immunization method used in this study caused significant damage to the health of the mice.

[0074] 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 trivalent HSV-2 mucosal vaccine composition based on the FliC / CpG adjuvant system, characterized in that, Including Salmonella typhimurium flagellar protein FliC, CpG oligonucleotide and HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein; The amino acid sequence of the flagellated protein FliC of Salmonella Typhimurium is shown in SEQ ID NO.1; The sequence of the CpG oligonucleotide is shown in SEQ ID NO.

3.

2. The HSV-2 trivalent mucosal vaccine composition based on the FliC / CpG adjuvant system according to claim 1, characterized in that, The mass ratio of the Salmonella Typhimurium flagellate protein FliC, CpG oligonucleotide, and HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein was 1:10:

15.

3. A method for preparing a trivalent HSV-2 mucosal vaccine based on a FliC / CpG adjuvant system, comprising the HSV-2 trivalent mucosal vaccine composition based on a FliC / CpG adjuvant system as described in claim 1 or 2, characterized in that, The HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system includes nasal drop vaccine and subcutaneous vaccine; The specific preparation method is as follows: HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, Salmonella typhimurium flagellate protein FliC, and CpG oligonucleotides were dissolved in physiological saline until the ratio of total volume to CpG oligonucleotide mass was 70 μL: 10 μg to obtain the nasal drop vaccine. HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, Salmonella typhimurium flagellate protein FliC, and CpG oligonucleotides were dissolved in physiological saline until the ratio of total volume to CpG oligonucleotide mass was 100 μL: 10 μg, thus obtaining the subcutaneous vaccine.

4. The method for preparing the HSV-2 trivalent mucosal vaccine based on the FliC / CpG adjuvant system according to claim 3, characterized in that, Dissolve 15 μg of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 1 μg of Salmonella typhimurium flagellate protein FliC, and 10 μg of CpG oligonucleotide in physiological saline to a total volume of 70 μL to obtain the nasal drop vaccine; each dose of nasal drop vaccine has a volume of 70 μL. Dissolve 15 μg HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 1 μg Salmonella typhimurium flagellate protein FliC, and 10 μg CpG oligonucleotide in physiological saline to a total volume of 100 μL to obtain a subcutaneous vaccine; each dose of subcutaneous vaccine has a volume of 100 μL.

5. An HSV-2 trivalent mucosal vaccine prepared by the method of the FliC / CpG adjuvant system as described in claim 3.

6. The use of the HSV-2 trivalent mucosal vaccine composition based on the FliC / CpG adjuvant system as described in claim 1 or 2 in the preparation of a vaccine for the prevention or treatment of diseases caused by infection with herpes simplex virus type 2.

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