A recombinant protein vaccine composition for preventing genital herpes and use thereof

Abstract

The present application relates to a kind of recombinant protein vaccine compositions for preventing genital herpes and its application, belong to biological medicine technical field.The vaccine composition includes antigen component and adjuvant component;Wherein, antigen component is composed of the extracellular domain of three proteins HSV-2 gB2, HSV-2 gD2, HSV-2 gE2;The adjuvant component is dmLT and MPLA.Animal experiment shows that the vaccine composition can induce high level of specific humoral immunity and mucosal immunity, significantly reduce acute phase genital tract viral load, reduce pathological damage, and can effectively block HSV-2 in dorsal root ganglion establish latent infection and inhibit latent period virus recurrence and discharge, with good biological safety and excellent protection effect, can be applied to the preparation of the drug for preventing HSV-2 infection and genital herpes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant protein vaccine composition for the prevention of genital herpes and its application. Background Technology

[0002] Herpes simplex virus type 2 (HSV-2) is the main pathogen causing genital herpes (GH). According to the World Health Organization, in 2016, approximately 491.5 million people aged 15-49 worldwide were infected with HSV-2, with a prevalence rate as high as 13.2%. HSV-2 infection is characterized by a single infection, lifelong latency, and recurrent infections. The virus can establish persistent latent infection in the dorsal root ganglion (DRG), and there is currently no eradication method or effective preventive vaccine.

[0003] Currently, there are several HSV-2 vaccine candidates on the market, mainly including monovalent or multivalent subunit vaccines targeting viral glycoproteins gB2, gC2, gD2, and gE2. Among these technologies, gB2 is primarily responsible for the fusion of the viral envelope with the host cell membrane; gC2 assists in the binding of the virus to receptors on the host cell surface and can bind complement C3b components to help the virus evade early innate immune clearance; gD2 is a key protein for viral receptor binding and is often used as a primary target for inducing neutralizing antibodies; gE2 mediates intercellular viral diffusion and blocks antibody-dependent cell-mediated cytotoxicity (ADCC) by binding to the Fc fragment of IgG.

[0004] However, existing vaccines still have many shortcomings. First, subunit vaccines targeting only gD2 failed to achieve ideal protective effects in phase III clinical trials, suggesting that a single-target strategy is insufficient to address the complex immune escape mechanisms of HSV-2, and optimization of multi-target combination strategies is urgently needed. Second, existing vaccines mainly induce humoral immune responses, lacking effective cellular immune activation, making it difficult to clear latent infections or prevent viral recurrence. Simultaneously, the lack of adjuvant systems that can strongly induce mucosal immunity leaves the initial immune barrier at the site of infection weak. These technical and strategic limitations restrict the clinical application effectiveness of existing vaccines, necessitating innovative solutions to improve overall immune protection.

[0005] In summary, despite numerous attempts to develop HSV-2 vaccines, due to limitations in immune response and the virus's high variability, an ideal vaccine that not only provides good immune protection but also overcomes the challenges of latent infection and recurrent infections has yet to be developed. This invention aims to overcome these shortcomings and provide a new technical approach for the immunization and control of HSV-2. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the prior art and provide a recombinant protein vaccine composition for the prevention of genital herpes and its application, thus providing a new technical approach for the immunization and prevention of HSV-2.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a recombinant protein vaccine composition for the prevention of genital herpes, comprising an antigen component and an adjuvant component; wherein the antigen component is composed of the extracellular domains of three proteins, HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2, and 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.3, and SEQ ID NO.4, respectively; the adjuvant component is dmLT and MPLA.

[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, dmLT, and MPLA was 10:10:10:10:25.

[0009] A second aspect of the present invention provides a method for preparing a recombinant protein vaccine for the prevention of genital herpes, using the aforementioned recombinant protein vaccine composition for the prevention of genital herpes, comprising 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 and the extracellular domains of HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 were obtained respectively and stored at -80℃. Step (4): The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 obtained in step (3) are thawed on ice. The MPLA adjuvant was dissolved in DMSO to prepare a 1 mg / mL MPLA stock solution; Dissolve the dmLT adjuvant in 1 mL of ultrapure water to obtain a dmLT stock solution of 3 mg / mL; Then, the extracellular domains of HSV-2 gB2 protein, HSV-2 gD2 protein, HSV-2 gE2 protein, MPLA stock solution, and dmLT stock solution were mixed and diluted with physiological saline to obtain a recombinant protein vaccine for the prevention of genital herpes.

[0010] Furthermore, each dose of the recombinant protein vaccine for the prevention of genital herpes contains 150 μL of HSV-2 gB2 protein extracellular domain, 10 μg of HSV-2 gD2 protein extracellular domain, 10 μg of HSV-2 gE2 protein extracellular domain, 10 μg of dmLT adjuvant, and 25 μg of MPLA adjuvant.

[0011] The third aspect of the present invention provides a recombinant protein vaccine for the prevention of genital herpes prepared by the method described above.

[0012] The fourth aspect of the present invention provides the use of the recombinant protein vaccine composition for the prevention of genital herpes in the preparation of a vaccine for the prevention or treatment of diseases caused by herpes simplex virus infection.

[0013] Furthermore, herpes simplex virus includes herpes simplex virus type 1 and herpes simplex virus type 2.

[0014] This invention develops a recombinant protein vaccine composition for the prevention of genital herpes, comprising an antigen component and an adjuvant component. The feasibility of using this vaccine for HSV-2 prevention was verified by evaluating its immunogenicity, safety, and protective effect after challenge in guinea pigs. This is expected to provide theoretical support for the development of HSV-2 vaccines.

[0015] The vaccine of this invention is administered via intramuscular injection, with a three-dose immunization schedule, each 14 days apart. This vaccine provides cross-protection against both HSV-2 and HSV-1 virus strains.

[0016] This invention is not a simple combination of adjuvants, but rather, based on the biological characteristics of HSV-2, which requires both "systemic immunity" and "mucosal barrier," it specifically selects dmLT (emphasizing mucosal response) and MPLA (emphasizing systemic response) for combination. Single adjuvants have inherent limitations in mechanism and cannot independently solve the specific technical problem of "completely blocking viral invasion and shedding."

[0017] Compared with the prior art, the beneficial effects of this invention are as follows: (1) This invention is the first to combine three key envelope glycoproteins of HSV-2, gB2, gD2 and gE2. These three antigens target different key stages of the viral infection cycle, forming a dual defense pattern of "external prevention of invasion and internal prevention of spread", thus achieving comprehensive blocking of HSV-2 infection.

[0018] (2) This invention is the first to use dmLT (double-mutant heat-labile enterotoxin) and MPLA (monophosphoryl ester A) in combination as a composite adjuvant for HSV-2 vaccines. dmLT is a highly effective mucosal adjuvant that can induce balanced Th1, Th2 and Th17 immune responses; MPLA is a TLR4 agonist that can enhance antiviral immune responses and promote antigen cross-presentation. The combination of the two produces a synergistic effect, significantly enhancing the immunogenicity of the vaccine.

[0019] (3) Histopathological evaluation showed that after immunization with the vaccine composition of the present invention, no obvious pathological damage was observed in the main organs of guinea pigs such as heart, liver, spleen, lungs and kidneys, demonstrating good biosafety.

[0020] (4) The present invention uses HSV-2 gB2, gD2 and gE2 proteins as antigens and dmLT and MPLA adjuvants as compound adjuvants to immunize guinea pigs by intramuscular injection, successfully inducing high levels of gB, gD and gE specific IgG antibodies and IgA antibodies, and effectively improving the neutralizing ability of immune serum against HSV-2 KM-1 virus strain and the cross-neutralizing ability against HSV-1 Strain17 virus strain.

[0021] (5) The genital herpes recombinant protein vaccine of the present invention induces a Th2-type humoral immune response.

[0022] (6) Experiments have shown that, compared with the negative control group (dmLT+MPLA), the gB2 / gD2 / gE2+dmLT+MPLA vaccine combination of the present invention exhibits multi-level protective effects after challenge: during the acute infection period, it significantly reduces the viral load in the vagina (about 1 log10) and alleviates pathological damage to the reproductive tract; during the latent infection period, it effectively inhibits viral recurrence and shedding, and blocks the establishment of latent infection by reducing the viral load in the dorsal root ganglion. Attached Figure Description

[0023] Figure 1 This is a graph showing the safety evaluation results of the vaccine in Example 2 of the present invention; where the scale bar is 500 μm. Figure 2Figure 3 shows the results of guinea pig serum-specific IgG detection in Example 3 of the present invention; wherein, Figures A, B, C, and D are the detection of specific IgG antibody levels of gB1, gC1, gD1, and gE1 antigens in serum, respectively; Figures E, F, G, and H are the detection of specific IgG antibody levels of gB2, gC2, gD2, and gE2 antigens in serum, respectively. Figure 3 Figure 3 shows the results of guinea pig serum-specific IgA detection in Example 3 of the present invention; wherein, Figures A, B, C, and D are the detection of specific IgA antibody levels of gB1, gC1, gD1, and gE1 antigens in serum, respectively; Figures E, F, G, and H are the detection of specific IgA antibody levels of gB2, gC2, gD2, and gE2 antigens in serum, respectively. Figure 4 Figure A shows the results of the guinea pig serum neutralization experiment in Example 3 of the present invention; Figure B shows the neutralizing antibody titer of HSV-1 strain 17 and the neutralizing antibody titer of HSV-2 strain KM-1. Figure 5 Figure 3 shows the ELISA results of cytokines in Example 3 of the present invention; wherein, Figure A shows the IL-2 concentration detection, Figure B shows the IL-4 concentration detection, Figure C shows the IL-10 concentration detection, and Figure D shows the IL-17 concentration detection. Figure 6 Figure 3 shows the results of mucosal humoral immune detection in Example 3 of the present invention; wherein, Figures A, B, C, and D show the detection levels of specific sIgA antibodies against gB1, gC1, gD1, and gE1 antigens in vaginal wash, respectively; Figures E, F, G, and H show the detection levels of specific sIgA antibodies against gB2, gC2, gD2, and gE2 antigens in vaginal wash, respectively. Figure 7 Figure 3 shows the results of mucosal humoral immunity detection in Example 3 of the present invention; wherein, Figures A, B, C, and D are the detection levels of specific IgG antibodies against gB1, gC1, gD1, and gE1 antigens in vaginal wash, respectively; Figures E, F, G, and H are the detection levels of specific IgG antibodies against gB2, gC2, gD2, and gE2 antigens in vaginal wash, respectively. Figure 8 This is a graph showing the survival rate of guinea pigs in each group during the acute phase after challenge in Example 4 of this invention. Figure 9 Figure 4 shows the results of the test of the protective effect against infection in the acute phase after challenge in Example 4 of the present invention; wherein, Figure A shows the percentage change in body weight of guinea pigs within 14 days of the acute phase after challenge, Figure B shows the average pathological score of the vagina of guinea pigs within 14 days of the acute phase after challenge, and Figure C shows the detection of viral load in vaginal wash fluid of guinea pigs within 14 days of the acute phase after challenge. Figure 10Figure A shows the results of the protective effect against relapse infection during the latent period after challenge in Example 4 of the present invention; Figure B shows the detection of viral relapse and shedding in the genitals during the relapse period after HSV-2 challenge infection in guinea pigs, and Figure B shows the results of viral latency detection in the dorsal root ganglia of guinea pigs on day 60 after HSV-2 challenge infection. In the figure, gB2 / gD2 / gE2 is labeled as the experimental group, gC2 / gD2 / gE2 is labeled as the positive control group, and dmLT+MPLA is labeled as the negative control group. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the embodiments.

[0025] 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.

[0026] Example 1: Preparation of recombinant protein antigen A recombinant protein vaccine composition for the prevention of genital herpes comprises HSV-2 recombinant protein and a compound adjuvant; the HSV-2 recombinant protein includes the extracellular domains of three proteins: gB2, gD2, and gE2, and the extracellular domains of three proteins: gC2, gD2, and gE2 are used as a positive control.

[0027] The amino acid sequences of the extracellular domains of HSV-2 gB2, gC2, gD2, and gE2 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The compound adjuvant consists of dmLT and MPLA adjuvants.

[0028] The amino acid sequence of the HSV-2 gB2 extracellular domain is as follows: GP67 signal peptide + gB2 (98-730 aa) + histidine tag + stop codon The amino acid sequence of the HSV-2 gC2 extracellular domain is as follows: GP67 signal peptide + gC2 (28-447aa) + histidine tag + stop codon The amino acid sequence of the extracellular domain of HSV-2 gD2 is as follows: GP67 signal peptide + gD2 (26-310 aa) + histidine tag + stop codon The amino acid sequence of the HSV-2 gE2 extracellular domain is as follows: GP67 signal peptide + gE2 (21-414aa) + histidine tag + stop codon Step (1), transformation of plasmids containing the gB2, gC2, gD2, and gE2 gene expression genes: The coding gene sequences corresponding to the extracellular domains of four proteins, HSV-2 gB2, HSV-2 gC2, HSV-2 gD2, and HSV-2 gE2, were cloned into the Hind III and EcoR I restriction sites of the pFastBac1 plasmid to obtain four corresponding recombinant pFastBac1 plasmids. Add 400 ng of each recombinant pFastBac1 plasmid to 100 μL of competent E. coli DH5α cells, mix well, incubate on ice for 30 min, then heat shock at 42°C for 45 s, quickly transfer to ice and incubate on ice for another 2 min, then add 500 μL of antibiotic-free SOC medium (Solepro: L1020-1L), shake at 37°C and 120 rpm for 1 h, take 100 μL of bacterial culture and spread it on solid medium containing ampicillin, and incubate upside down at 37°C for 12 h.

[0029] 200mL of ampicillin-resistant solid culture medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 4g agar powder, 20mg ampicillin, 200mL water.

[0030] 200mL ampicillin liquid culture medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 20mg ampicillin, 200mL water.

[0031] 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, and finally recombinant pFastBac1gB2 plasmid DNA, recombinant pFastBac1gC2 plasmid DNA, recombinant pFastBac1gD2 plasmid DNA, and recombinant pFastBac1gE2 plasmid DNA were obtained.

[0032] Step (2), preparation of recombinant Bacmid DNA: Recombinant pFastBac1gB2, pFastBac1gC2, pFastBac1gD2, and pFastBac1gE2 plasmid DNAs containing the target gene, which were correctly sequenced, were extracted and transformed into MAXEfficiency™ DH10Bac™ (Thermo Fisher: 2498470A) competent cells to transpose into Bacmid plasmids. Colonies containing the recombinant Bacmid plasmids were screened and identified using blue-white screening.

[0033] The specific method for preparing recombinant Bacmid DNA is as follows: 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 pFastBac1gC2 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 antibiotic-free 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 and 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, gC2 recombinant Bacmid DNA, gD2 recombinant Bacmid DNA, and gE2 recombinant Bacmid DNA.

[0034] 200 mL of solid selective medium containing four resistances: 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, 200 mL water; 200 mL solid selective medium: 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, 200 mL water; 200mL liquid selective medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 50μg / mL kanamycin, 7μg / mL gentamicin, 10μg / mL tetracycline, 200mL water.

[0035] Step (3), viral amplification and purification: 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), they were cultured. 6 When the cell viability is >95%, the cells are increased to 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 thoroughly by inverting, and incubated at room temperature for 5 min. Then, 1 μg / well of gB2 recombinant Bacmid DNA, gC2 recombinant Bacmid DNA, gD2 recombinant Bacmid DNA, and gE2 recombinant Bacmid DNA were added to the diluted transfection reagent, mixed thoroughly by inverting, and incubated at room temperature for 5 min to obtain four mixtures. Finally, each mixture was added to sf9 cells and cultured at 27℃ for 72-96 h until the sf9 cells exhibited obvious pathological changes such as rounding, swelling, budding, and breakage. The supernatant was collected by centrifugation at 300g for 5 min and used as the P0 generation virus stock solution.

[0036] Then take 20-80 μL of P0 generation virus stock solution 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.

[0037] 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.

[0038] After centrifugation, the culture 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 for 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 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 (with BSA as a control). The extracellular domains of HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 were obtained with a purity of over 90%. The obtained proteins were dissolved in protein storage buffer and stored at -80°C. The protein storage buffer formulation was: 50 mM Tris-HCl, 500 mM NaCl, 5% (v / v) glycerol, pH 8.0.

[0039] Step (4), preparation of vaccine injection: The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2 obtained from step (3) were thawed on ice. The MPLA adjuvant was dissolved in DMSO to prepare a 1 mg / mL MPLA stock solution; Dissolve the dmLT adjuvant in 1 mL of ultrapure water to obtain a dmLT stock solution of 3 mg / mL; Then, the extracellular domains of HSV-2 gB2 protein, HSV-2 gD2 protein, HSV-2 gE2 protein, MPLA stock solution, and dmLT stock solution were mixed and diluted with physiological saline to obtain a recombinant protein vaccine for the prevention of genital herpes.

[0040] Each dose of the recombinant protein vaccine for the prevention of genital herpes contains 150 μL of HSV-2 gB2 protein extracellular domain, 10 μg of HSV-2 gD2 protein extracellular domain, 10 μg of HSV-2 gE2 protein extracellular domain, 10 μg of dmLT adjuvant, and 25 μg of MPLA adjuvant.

[0041] Example 2: Vaccine combination preparation and safety evaluation 2.1 Vaccine preparation: We immunized guinea pigs with a mixture of purified HSV-2 recombinant protein and adjuvant. The guinea pigs used were purchased from Vital River Company, all of which were female and weighed about 300g at 6 weeks of age. The guinea pig immunization experiment was set up with experimental group, positive control group and negative control group; the experimental group was injected with gB2 / gD2 / gE2+dmLT+MPLA; the positive control group was injected with gC2 / gD2 / gE2+dmLT+MPLA, and the negative control group was injected with dmLT+MPLA; each guinea pig was immunized with 10μg of each protein extracellular domain, 25μg of dmLT and 10μg of MPLA, with 12 guinea pigs in each group, by injection into the posterolateral thigh muscle, with each immunization dose of 150μL / guinea pig, and the vaccine was prepared according to the method of step (4) above.

[0042] 2.2 Safety Evaluation Fourteen days after the last immunization, some animals were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were collected for HE staining and pathological examination. The results showed that the structures of all major organs were intact, with no obvious edema, degeneration, necrosis, or inflammatory cell infiltration. Figure 1 This indicates that the vaccine composition of the present invention has good biosafety.

[0043] Example 3: Evaluation of vaccine immunogenicity 3.1 Detection of serum-specific antibodies IgG and IgA: To determine the level of specific antibodies in guinea pig serum after three doses of vaccine immunization, this invention uses enzyme-linked immunosorbent assay (ELISA) to detect specific IgG and IgA antibodies against gB, gC, gD, and gE proteins in guinea pig serum.

[0044] Figure 2 (Serium IgG detection) results showed that no specific IgG antibodies were detected in the negative control group (dmLT+MPLA). Both the positive control group (gC2 / gD2 / gE2) and the experimental group (gB2 / gD2 / gE2) induced high levels of HSV-1 and HSV-2 related antigen-specific IgG antibody responses. Figure 2 A- Figure 2 The results showed that the HSV-1 gB-specific IgG level induced in the experimental group was significantly higher than that in the positive control; while gC and gD were significantly lower than those in the positive control group; however, gD did not differ significantly from the positive control group. Figure 2 E- Figure 2 The F-values ​​in the experimental group showed that the HSV-2 gB and gC-specific IgG induced by the experimental group were significantly higher than those in the positive control group. Figure 2 G- Figure 2 The H in the data showed that the gD and gE experimental groups were comparable to the positive control group in terms of the level of induced specific IgG.

[0045] Figure 3(Serium IgA detection) results showed that no specific IgA antibodies were detected in the negative control group. Both the positive control group and the experimental group could induce HSV-1 and HSV-2 related antigen-specific IgA antibody responses. Figure 3 The A in the figure indicates that the experimental group showed a significantly higher induction of HSV-1 gB-specific IgA compared to the positive control group; Figure 3 B and Figure 3 The D-values ​​in the study showed that there was no significant difference in the induction levels of HSV-1 gC and gE-specific IgA between the two groups; Figure 3 The C value indicates that the HSV-1 gD-specific IgA induced in the positive control group was significantly higher than that in the experimental group. Figure 3 E and Figure 3 The H in the data indicates that the experimental group had comparable induction of specific IgA for HSV-2 gB and gE to the positive control group. Figure 3 F and Figure 3 The results showed that the induction of HSV-2 gC and gD specific IgA in the positive control group was significantly higher than that in the experimental group.

[0046] comprehensive Figure 2 and Figure 3 The results showed that the vaccine composition of the present invention can induce a strong humoral immune response against HSV-1 and HSV-2 related antigens. The experimental group produced levels of specific IgG antibodies comparable to or higher than those in the positive control group.

[0047] 3.2 Serum neutralizing antibody titer determination The titer of neutralizing antibodies in serum was detected using a serum neutralization assay, and the results were calculated using the Reed-Muench method. The results are as follows: Figure 4 As shown, no significant neutralizing activity was observed in the negative control group (dmLT+MPLA). For HSV-1 strain 17 ( Figure 4 The neutralizing antibody titers in the A group and the positive control group (gC2 / gD2 / gE2) were approximately 1.0 log. 10 The neutralizing antibody titers in the experimental group (gB2 / gD2 / gE2) were slightly lower than those in the positive control group, indicating that the vaccine group of this invention has a certain neutralizing ability against HSV-1 strain 17. For the HSV-2 clinical isolate KM-1 ( Figure 4 In the B group, the neutralizing antibody titer in the positive control group was approximately 1.5 log₂(b). 10 The neutralizing antibody titer in the experimental group was approximately 1.0 log. 10 This indicates that the vaccine composition of the present invention has a significant neutralizing effect on the HSV-2 clinical isolate KM-1. The above results demonstrate that the vaccine composition of the present invention can induce the production of neutralizing antibodies against HSV-2 and has cross-neutralizing activity against HSV-1, exhibiting good immunization efficacy.

[0048] 3.3 Cytokine Level Detection To detect the Th1 and Th2 responses of guinea pigs induced by the vaccine of this invention, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of Th1 cytokines IFN-γ (Sandwich ELISA; LSBio LS-F5319) and IL-2 (Sandwich ELISA; LSBio LS-F5967) and Th2 cytokines IL-4 (Biorbyt orb561589) and IL-10 (Sandwich ELISA; LSBio LS-F5696). We also detected the response of mucosal adjuvants to induce IL-17 (Krishgen BioSystems; KLY0097) mucosal immune responses in guinea pigs. Spleen cells were isolated on day 14 after the last immunization. The prepared gB2, gC2, gD2, and gE2 proteins were used to stimulate the spleen cells at a stimulation concentration of 2 μg / mL per well, and the secretion of each cytokine was detected. Detailed procedures are described in the respective kit instructions. Results are as follows: Figure 5 As shown: The results indicate that the levels of IFN-γ in the culture supernatant of each group were below the detection limit (results not shown), and IL-2 ( Figure 5 A in IL-10 Figure 5 C) and IL-17 ( Figure 5 There was no significant difference in IL-4 levels in the experimental group (gB2 / gD2 / gE2) compared to the negative and positive control groups. Figure 5 The significantly increased secretion level of IL-4 (B) in the vaccine composition (P<0.01) indicates that the vaccine composition of the present invention can more effectively induce IL-4-related immune responses. These results demonstrate that the vaccine composition of the present invention can induce Th2-biased cytokine responses and exhibits good immunogenicity.

[0049] 3.4 Evaluation of mucosal humoral immunity Since genital herpes infection originates in the genital tract mucosa, establishing an effective mucosal immune barrier is crucial for preventing HSV-2 infection. This experiment used a recombinant subunit vaccine combined with a novel composite mucosal adjuvant, dmLT+MPLA, administered via intramuscular injection, aiming to induce both systemic and mucosal immunity. To this end, vaginal swabs were collected from guinea pigs in the first and second weeks after three booster immunizations, and the levels of specific sIgA and IgG antibodies in vaginal secretions were detected using ELISA.

[0050] Figure 6(Vaginal swab sIgA test) results showed that, compared with the negative control group (dmLT+MPLA), both the positive control group (gC2 / gD2 / gE2) and the experimental group (gB2 / gD2 / gE2) showed varying degrees of elevated sIgA levels. Figure 6 A- Figure 6 B in the figure indicates that the experimental group had a more prominent gB1-specific sIgA level, which was slightly higher than that of the positive control group, while the gC1-specific sIgA level was slightly lower than that of the positive control group. Figure 6 C- Figure 6 The results showed that there was no significant difference in gD1-specific sIgA levels between the experimental group and the positive control group, but the gE1-specific sIgA levels in the experimental group were lower than those in the positive control group. Figure 6 E- Figure 6 The F-values ​​in the study showed that the experimental group had a significantly higher level of gB2-specific sIgA than the positive control group, while the gC2-specific sIgA levels were similar between the two groups. Figure 6 G- Figure 6 The H in the data indicates that the experimental group and the positive control group were basically equivalent in terms of the specific sIgA levels of gD2 and gE2 of HSV-2.

[0051] Figure 7 (Vaginal swab IgG test) results showed that the overall level in the negative control group was low; both the positive control group and the experimental group could induce a significant IgG response. Figure 7 A- Figure 7 B in the figure indicates that, compared with the positive control, the experimental group had a higher ability to induce gB1-specific IgG, while the gC1-specific IgG was similar in both groups; Figure 7 C- Figure 7 The results showed that there were no significant differences in gD1 and gE1-specific IgG between the experimental group and the positive control group; Figure 7 E- Figure 7 The F-values ​​in the data show that the experimental group showed a significant increase in gB2-specific IgG and a significant decrease in gC2-specific IgG. Figure 7 G- Figure 7 The H in the data indicates that both the experimental group and the positive control group showed high and similar levels of gD2 and gE2-specific IgG.

[0052] comprehensive Figure 6 and Figure 7 The results showed that the vaccine composition of the present invention can induce specific sIgA and IgG antibody responses locally in the reproductive tract mucosa, exhibiting good mucosal immunogenicity and certain cross-immunity potential.

[0053] Example 4: Protective effect of vaccine against viral challenge 4.1 Virus Challenge Experiment Protective efficacy evaluation is an important indicator in vaccine evaluation. To evaluate the protective effect of several vaccines against HSV-2 virus, we used the 15-LD50 of HSV-2 KM-1. 50 (6.15×10) 5 The mice were challenged vaginally with a dose of PFU / mouse to simulate their natural infection state. Various physiological indicators (body weight, survival rate, and reproductive tract lesion score; vaginal swabs were collected every other day to detect viral load) were monitored from day 1 post-challenge until day 14, two weeks post-challenge.

[0054] 4.2 Evaluation of survival rate after virus challenge To evaluate the protective effect of each immunization group, animals were challenged with HSV-2 KM-1 strain on day 14 after the last immunization, and the survival of animals in each group was continuously observed after challenge. The results are as follows: Figure 8 As shown in the figure, animals in the negative control group (dmLT+MPLA) began to die on day 9 after viral challenge, and the survival rate dropped to approximately 50% by day 11, remaining at the observation endpoint. In contrast, animals in the positive control group (gC2 / gD2 / gE2) did not die throughout the entire observation period, maintaining a survival rate of 100%. Animals in the experimental group (gB2 / gD2 / gE2) also did not die throughout the entire observation period after viral challenge, with a survival rate of 100%, demonstrating complete protection against viral challenge. These results indicate that the vaccine composition of the present invention can provide significant protection to immunized animals after viral challenge, improving animal survival rates. Its protective effect is comparable to that of the positive control group and significantly superior to that of the negative control group, demonstrating that the vaccine composition of the present invention has good immunoprotective efficacy.

[0055] 4.3 Clinical symptoms in the acute phase after virus challenge After challenge, daily weight changes in each group of animals were measured, vaginal lesions were scored, and vaginal douches were collected every other day. Viral load was detected by qPCR. Primer sequences: Forward primer F: 5'-aacacatccccctgttctgg-3' (SEQ ID NO. 9); Reverse primer R: 5'-cgaccagacaaacgaacgc-3' (SEQ ID NO. 10); Probe: 5'-FAM-Agcaccaccatccacacggc-BHQ1-3' (SEQ ID NO. 11); The reaction system was 10 μL, containing 4 μL template DNA; 0.4 μL each of the forward and reverse primers (both at 0.4 μM); 0.2 μL of the probe (at 0.2 μM); and 5 μL of 2×AceQUniversal U+ Probe Master Mix (Novizan: Q513-02). The reaction conditions were as follows: incubation at 37°C for 2 min; pre-denaturation at 95°C for 5 min; and then 45 cycles, each cycle including denaturation at 95°C for 10 s, annealing at 60°C for 30 s.

[0056] The results are as follows Figure 9 As shown. Figure 9 A in the study showed that the body weight of animals in the negative control group (dmLT+MPLA) decreased slightly after the challenge, while the body weight of animals in the positive control group (gC2 / gD2 / gE2) and the experimental group (gB2 / gD2 / gE2) remained stable overall, with the experimental group showing a certain upward trend in the later stage. Figure 9 B in the study showed that the negative control group developed more obvious vaginal lesions after the challenge, and the lesion score gradually increased over time; the lesion severity in the positive control group was less severe than that in the negative control group; the experimental group had a lower overall lesion score and showed milder local lesion manifestations during the observation period. Figure 9 The results in Figure C show that the viral load in the vaginal rinsing fluid of all three groups of animals gradually decreased with the progress of infection. The experimental group showed a lower viral load and a faster rate of decrease in the early stage of challenge, suggesting a good inhibitory effect on viral replication. These results indicate that the vaccine composition of this invention can alleviate clinical symptoms during the acute infection phase, reduce the severity of local lesions, and promote viral clearance after viral challenge, demonstrating a good immunoprotective effect.

[0057] 4.4 Evaluation of latent infection To evaluate viral recurrence during the incubation period after infection in each group of animals, vaginal swabs were collected for 20 consecutive days from day 29 to day 49 post-infection, and the viral load (copies / mg) in the dorsal root ganglion samples was measured at the endpoint (day 60 after infection). Results are as follows: Figure 10 As shown, Figure 10As shown in Figure A: In the negative control group (dmLT+MPLA), one animal tested positive for viral load during the two-day incubation period, with viral load ranging from 1000 to 10000 copies / μL on one day and from 100 to 1000 copies / μL on the other day; the remaining animals were negative. In the positive control group (gC2 / gD2 / gE2), only one animal showed a low-level positive result on one day, with viral load ranging from 100 to 1000 copies / μL; the remaining animals were negative. Similarly, in the experimental group (gB2 / gD2 / gE2), only one animal showed a low-level positive result on one day; the remaining animals were negative. Figure 10 As shown in Figure B: the viral load in the dorsal root ganglia of each group was generally low, with most individuals approaching the detection limit (dashed line). Furthermore, the overall viral load in the vaccine-immunized group was low, suggesting that immunization can reduce viral persistence in the ganglia to some extent, thereby reducing the establishment of latent infection. These results indicate that the vaccine composition described in this invention can reduce recurrent viral shedding during the latent period after infection, demonstrating excellent defensive functions in both limiting viral entry into the ganglia and inhibiting reactivation, making it a potential candidate vaccine composition for preventing HSV-2 infection.

[0058] 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 recombinant protein vaccine composition for the prevention of genital herpes, characterized in that, It includes an antigen component and an adjuvant component; wherein the antigen component is composed of the extracellular domains of three proteins, HSV-2 gB2, HSV-2 gD2, and HSV-2 gE2, and 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.3, and SEQ ID NO.4, respectively; the adjuvant component is dmLT and MPLA.

2. The recombinant protein vaccine composition for the prevention of genital herpes according to claim 1, characterized in that, The mass ratio of HSV-2gB2 protein extracellular domain, HSV-2 gD2 protein extracellular domain, HSV-2 gE2 protein extracellular domain, dmLT, and MPLA is 10:10:10:10:

25.

3. A method for preparing a recombinant protein vaccine for the prevention of genital herpes, comprising the recombinant protein vaccine composition for the prevention of genital herpes as described in claim 1 or 2, 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 and the extracellular domains of HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 were obtained respectively and stored at -80℃. Step (4): The extracellular domains of the three proteins HSV-2 gB2, HSV-2 gD2 and HSV-2 gE2 obtained in step (3) are thawed on ice. The MPLA adjuvant was dissolved in DMSO to prepare a 1 mg / mL MPLA stock solution; Dissolve the dmLT adjuvant in 1 mL of ultrapure water to obtain a dmLT stock solution of 3 mg / mL; Then, the extracellular domains of HSV-2 gB2 protein, HSV-2 gD2 protein, HSV-2 gE2 protein, MPLA stock solution, and dmLT stock solution were mixed and diluted with physiological saline to obtain a recombinant protein vaccine for the prevention of genital herpes.

4. The method for preparing the recombinant protein vaccine for the prevention of genital herpes according to claim 3, characterized in that, Each dose of the recombinant protein vaccine for the prevention of genital herpes contains 150 μL of HSV-2 gB2 protein extracellular domain, 10 μg of HSV-2 gD2 protein extracellular domain, 10 μg of HSV-2 gE2 protein extracellular domain, 10 μg of dmLT adjuvant, and 25 μg of MPLA adjuvant.

5. The recombinant protein vaccine for preventing genital herpes prepared by the method for preparing the recombinant protein vaccine for preventing genital herpes according to claim 3.

6. The use of the recombinant protein vaccine composition for the prevention of genital herpes according to claim 1 or 2 in the preparation of a vaccine for the prevention or treatment of diseases caused by herpes simplex virus infection.

7. The use of the recombinant protein vaccine composition for preventing genital herpes according to claim 6 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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