Hsv-2 tri-antigenic tandem recombinant protein vaccine composition and application thereof

Abstract

The present application relates to a kind of HSV-2 three antigen tandem recombinant protein vaccine compositions and its application, belong to the field of biotechnology.The vaccine composition includes antigen and composite adjuvant, antigen is HSV-2 gC2-gD2-gE2 three antigen tandem recombinant protein, composite adjuvant is CpG oligonucleotide and aluminum adjuvant;Wherein, HSV-2 gC2-gD2-gE2 three antigen tandem recombinant protein amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2.The present application greatly reduces the time cost and economic cost in the process of protein preparation, compared with the original trivalent combination vaccine, it can induce the specific antibody level and neutralizing virus ability of mouse generation does not occur reduction, which provides a kind of new idea for developing advanced multi-target antigen HSV-2 virus vaccine, also provides a kind of safe and effective HSV-2 virus candidate vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an HSV-2 triantigen tandem recombinant protein vaccine composition and its application. Background Technology

[0002] Herpesviruses (Herpesviridae) are a family of DNA viruses. The main members of the herpesvirus family include herpes simplex virus type 1 (Herpes simplex 1), herpes simplex virus type 2 (Herpes simplex 2), and herpes zoster virus (Herpes zoster), which mainly cause diffuse skin lesions.

[0003] Herpes simplex virus consists of a genome, a capsid, and an envelope. The genome is a long, double-stranded linear DNA molecule encased in an icosahedral (T=16) protein capsid, which is then surrounded by an envelope. The genome contains at least 74 genes that encode a variety of proteins involved in the formation of the viral capsid and envelope, as well as controlling viral replication and infectivity.

[0004] During primary infection, herpes simplex virus replicates and releases itself in the surrounding mucosal and skin tissues, spreading from infected epithelial cells to the axons of sensory neurons at the site of primary infection, and then retrogradely translocating to their respective dorsal root ganglia. Recurrent infection stems from the reactivation of neurons, followed by viral replication and anterograde transport to cells in the surrounding areas innervated by their respective neurons. Among the 12 glycoproteins in the HSV-2 envelope, gB, gD, and gH / gL heterodimers play important roles in extracellular viral entry and intercellular diffusion. Studies have shown that expression of only these four glycoproteins can promote cell fusion. gD specifically binds to one of three entry receptors—herpesvirus entry mediator (HVEM), connexin 1, or 3-O heparan sulfate—promoting a conformational change in gD and interacting with viral surface glycoproteins H and L. The three gH / gL heterodimers stabilize the conformation of the gB protein, thereby exposing the gB protein and allowing it to bind to the cell surface receptor glycosaminoglycan to form a complex, creating an entry pore in the viral capsid, allowing the viral contents to enter the cell.

[0005] Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are chronic, highly prevalent viral infections causing significant morbidity worldwide. HSV-2 is sexually transmitted and is a leading cause of genital ulcer disease (GUD). HSV-1 can also be transmitted to the genital tract through oral-genital contact. A significant proportion of genital HSV infections go unrecognized clinically, although the infection can still be transmitted even when asymptomatic, contributing to the high global prevalence. Globally, the incidence is disproportionately high among women. HSV-2 also increases the risk of HIV infection and transmission, thus contributing to HIV morbidity and prevalence. Currently, there is no marketed HSV-2 vaccine. The main treatments are acyclovir, valacyclovir, and famciclovir, which can alleviate symptoms but do not eliminate the virus. While anti-HSV-2 drugs can suppress viral replication and maintain the virus in the latent period, they cannot completely eradicate the virus or prevent frequent reactivation. Therefore, there is an urgent need to develop safe, effective, and novel drugs and combination therapies. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an HSV-2 triantigen tandem recombinant protein vaccine composition and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of this invention provides an HSV-2 triantigen tandem recombinant protein vaccine composition, comprising an antigen and a compound adjuvant, wherein the antigen is an HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, and the compound adjuvant is a CpG oligonucleotide and an aluminum adjuvant; wherein the amino acid sequence of the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0009] Furthermore, the mass ratio of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, CpG oligonucleotide, and aluminum adjuvant is 15:50:125.

[0010] A second aspect of the present invention provides a method for preparing an HSV-2 triantigen tandem recombinant protein vaccine, using the above-mentioned HSV-2 triantigen tandem recombinant protein vaccine composition, characterized by comprising the following steps:

[0011] After thawing the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein solution dissolved in protein storage buffer on ice, mix the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein with aluminum adjuvant, and let it stand at 4°C with gentle shaking overnight. Before vaccination, mix it with CpG oligonucleotides to obtain the HSV-2 triantigen tandem recombinant protein vaccine.

[0012] Furthermore, the protein storage buffer consists of 50 mM Tris-HCl, 500 mM NaCl, 5% glycerol, and pH 8.0.

[0013] Furthermore, when shaking overnight, the speed is 80 rpm.

[0014] The third aspect of the present invention provides an HSV-2 triantigen tandem recombinant protein vaccine prepared by the above-mentioned method for preparing the HSV-2 triantigen tandem recombinant protein vaccine.

[0015] The fourth aspect of the present invention provides the use of the above-described HSV-2 triantigen tandem recombinant protein vaccine composition in the preparation of medicaments for the prevention and / or treatment of HSV-2.

[0016] In this invention, the extracellular segment of the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein is selected. The amino acid sequences of three independent proteins (HSV-2 gC2, gD2, and gE2) are tandemly fused using a short peptide linker sequence (GGGGS) consisting of four glutamic acid residues and one serine residue to obtain a triantigen tandem recombinant protein HSV-2 gC2-gD2-gE2. A GP67 secretion signal peptide sequence and six histidine tags are inserted at the N-terminus of the amino acid sequence of this triantigen tandem recombinant protein. The purpose is to more efficiently induce the expression of secretory proteins in Sf9 cells and to purify the required antigen proteins in cell cultures.

[0017] In this invention, the HSV-2 triantigen tandem recombinant protein vaccine composition, comprising the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, is prepared using a baculovirus-insect cell expression system. The specific preparation method is as follows:

[0018] Step (1): The coding gene sequences corresponding to the extracellular domains of the three proteins HSV-2 gC2-gD2-gE2 were tandemly docked and synthesized by Yunnan Genscript Biotech Co., Ltd. Then, the obtained HSV-2 gC2-gD2-gE2 tandem protein gene sequence was cloned into the Hind III and EcoRI restriction sites of the pFastBac1 plasmid to obtain the recombinant HSV-2 gC-gD2-gE2pFastBac1 plasmid;

[0019] Step (2): After verifying the recombinant HSV-2 gC-gD2-gE2 pFastBac1 plasmid by double digestion with Hind III and EcoR I and gene sequencing, the HSV-2 gC-gD2-gE2 pFastBac1 plasmid with the correct gene sequence was transfected into DH10Bac competent cells containing the Bacmid plasmid for plasmid recombination. The recombinant Bacmid plasmid carrying the HSV-2 gC2-gD2-gE2 gene sequence was successfully recombined by blue-white screening.

[0020] Step (3): The recombinant Bacmid plasmid carrying the HSV-2 gC2-gD2-gE2 gene sequence from step (2) was transfected into Sf9 cells for culture to obtain HSV-2 gC2-gD2-gE2 P1 generation reconstituted baculovirus. The cells were then passaged in Sf9 cells to the P3 generation. The P3 generation HSV-2 gC2-gD2-gE2 recombinant baculovirus was cultured in 4L Sf9 cells to express a large amount of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein. After purifying the protein in the cell culture supernatant by nickel column, HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein with a purity higher than 90% was obtained and stored at -80°C in protein storage buffer (50mM Tris-HCl, 500mM NaCl, 5% glycerol, pH 8.0).

[0021] When using the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein vaccine composition of the present invention, the dosage for each injection for mouse immunization is: 15 μg HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein, 50 μg CpG oligonucleotide, and 125 μg aluminum adjuvant.

[0022] In this invention, when preparing the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein vaccine composition, the protein antigen solution is thawed on ice, the required amount is taken and mixed evenly with the required amount of aluminum adjuvant, and placed in a 4°C refrigerator on a rotating rack and slowly shaken overnight to ensure sufficient adhesion between the protein antigen and the aluminum adjuvant. Before vaccination, it is then mixed evenly with CpG oligonucleotides.

[0023] In this invention, the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein vaccine composition is administered via intramuscular injection, with three doses given two weeks apart. It has application value in the prevention and treatment of HSV-2.

[0024] This invention upgrades the protein structure and preparation process of existing HSV-2 gC2, gD2, and gE2 trivalent protein vaccines, significantly reducing the time and economic costs associated with protein preparation. Furthermore, after tandemly fusing the three proteins, the vaccine of this invention does not exhibit a decrease in the level of specific antibodies induced in mice or its ability to neutralize the virus compared to the original trivalent combination vaccine; rather, it is comparable to the level of a single antigen combination of HSV-2 gC2, gD2, and gE2. This provides a novel approach for developing advanced multi-target antigen HSV-2 virus vaccines and also offers a safe and effective HSV-2 virus candidate vaccine.

[0025] Compared with the prior art, the beneficial effects of this invention are as follows:

[0026] (1) Improved protein expression efficiency. In the prior art, the three proteins HSV-2 gC2, gD2 and gE2 need to be expressed separately in different expression tanks. In this invention, the three proteins can be expressed simultaneously in one expression tank.

[0027] (2) The HSV-2 gC2-gD2-gE2 of the present invention maintains immunogenicity comparable to that of the prior art after the three single proteins are expressed in tandem;

[0028] (3) It reduces the quality control costs and safety risks in the production process. This invention only requires quality control evaluation of one protein product, thereby avoiding the tedious task of reviewing other protein products when one of the protein products fails to meet the quality control standards during the quality control evaluation of multiple protein products. Attached Figure Description

[0029] Figure 1 The values ​​represent the levels of specific IgG antibodies in mouse serum after three immunizations; where A, B, and C represent the levels of specific IgG antibodies against gC2, gD2, and gE2 proteins in mouse serum, respectively; Blank represents the blank control well.

[0030] Figure 2 The values ​​represent the levels of specific IgG subtype antibodies in mouse serum after three immunizations; where A: reflects the level of specific IgG1 antibody in mouse serum; B: reflects the level of specific IgG2a antibody in mouse serum; and C: reflects the difference between the levels of specific IgG1 and IgG2a antibodies in mouse serum.

[0031] Figure 3 The titers of neutralizing antibodies in mouse serum after three immunizations are shown; where A: reflects the titer of neutralizing antibodies against HSV-2 G strain in mouse serum; B: reflects the titer of neutralizing antibodies against HSV-1 17 strain in mouse serum.

[0032] Figure 4The image shows the ELISPOT assay for mouse-specific T cell responses after three immunizations. A: Reflects the responsiveness of mouse IFN-γ+ specific T cells; B: Reflects the responsiveness of mouse IL-4+ specific T cells; C: Partial original ELISPOT image.

[0033] Figure 5 The values ​​represent the cytokine content in the supernatant of mouse spleen cell culture after three immunizations; where A and B reflect the concentrations of Th1 cytokines IFN-γ and IL-2 in the supernatant of mouse spleen cell culture; and C and D reflect the concentrations of Th2 cytokines IL-4 and IL-10 in the supernatant of mouse spleen cell culture.

[0034] Where: ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the embodiments.

[0036] 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.

[0037] Unless otherwise stated, percentages in this invention are mass percentages.

[0038] 1. Gene sequence origin and composition:

[0039] The triantigen tandem recombinant protein (HSV-2 gC2-gD2-gE2) in the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein vaccine composition of the present invention is synthesized by selecting the extracellular domain, and the gene sequence is synthesized after codon optimization by Kunming Genscript Biotech Co., Ltd. and ligated into the pFastBac1 vector. The synthesized gene includes an enzyme restriction site, a signal peptide, a target gene, and a stop codon, and its full-length sequence is obtained. Its amino acid sequence is shown in SEQ ID NO.1 and its nucleotide sequence is shown in SEQ ID NO.2.

[0040] 2. Construction of expression plasmid containing HSV-2 gC2-gD2-gE2 gene:

[0041] (1) Take 400 ng of pFastBac1 plasmid containing HSV-2 gC2-gD2-gE2 provided by Kunming Genscript Biotech Co., Ltd. and add it to 500 μL of competent DH5α cells. Mix well, incubate on ice for 30 min, heat shock at 42℃ 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℃ and 120 rpm for 1 h. Take 100 μL of bacterial solution and spread it on ampicillin resistant (Amp+) LB solid medium. Incubate upside down at 37℃ for 12 h.

[0042] Single colonies were picked and inoculated into 10 mL of LB (Amp+) liquid medium and cultured at 37°C and 250 rpm for 8 h. 1 mL of the bacterial culture was sent to a sequencing company for sequencing identification. 10 μL of the remaining bacterial culture was inoculated into 200 mL of LB (Amp+) liquid medium and cultured at 37°C and 250 rpm for 16 h. The plasmid was then extracted using the PureYield plasmid Maxiprep system plasmid extraction kit (Promega: A2393) to obtain a large number of complete HSV-2 gC2-gD2-gE2 pFastBac1 plasmids.

[0043] The formulation of 200mL ampicillin-resistant LB (Amp+) solid medium is as follows: 2g tryptone, 1g yeast extract, 2g sodium chloride, 4g agar powder, 20mg ampicillin, and 200mL ultrapure water.

[0044] The formula for 200mL LB (Amp+) liquid culture medium is: 2g tryptone, 1g yeast extract, 2g sodium chloride, 20mg ampicillin, and 200mL ultrapure water.

[0045] (2) Further construction of recombinant baculovirus plasmid (Bacmid plasmid): 300 ng of the HSV-2 gC2-gD2-gE2 pFastBac1 plasmid obtained above was transfected into 50 μL of DH10Bac competent cells containing the Bacmid plasmid [MAXEfficiency™ DH 10 Bac™ (Thermo Fisher: 2498470A)]. Utilizing the characteristic of the Bacmid plasmid to homologously recombine exogenous genes into its own plasmid, the HSV-2 gC2-gD2-gE2 gene sequence on the pFastBac1 plasmid was recombined and integrated into the Bacmid plasmid. The operation steps are shown in (3).

[0046] (3) Blue-white spot selection to identify colonies containing recombinant rod-like particles: Prepare LB selective agar plates in advance and preheat them to 37℃ before use. Take 300ng of the pre-prepared HSV-2 gC2-gD2-gE2 pFastBac1 plasmid and add it to 50μL of competent DH10Bac bacteria. Mix gently, incubate on ice for 30min, then heat shock at 42℃ for 45s, quickly transfer to ice and incubate on ice for another 2min; then add 950μL of antibiotic-free SOC medium (Solepro: L1020-1L), and incubate at 37℃ and 225rpm for 4h. After the incubation period, use antibiotic-free SOC medium to test the bacterial culture for 10 hours. -1 10 -2 10 -3 Serial dilutions were performed, with 100 μL of each dilution added to LB selective agar plates preheated to 37°C. After even spreading, the plates were incubated upside down at 37°C for 48 h. At this point, blue and white colonies appeared. Isolated white positive colonies were picked and streaked onto LB selective agar plates to ensure that all newly grown colonies were white positive colonies. Single colonies were picked and inoculated into 10 mL of LB (TC+, K+, GM+) liquid medium and incubated 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 50 mL of LB (TC+, K+, GM+) liquid medium and incubated at 37°C and 250 rpm for 16 h. The plasmid was extracted using the Beyotime brand baculovirus shuttle vector Bacmid mini-extraction kit (Beyotime: D0031) to obtain a recombinant Bacmid plasmid containing the HSV-2 gC2-gD2-gE2 gene sequence.

[0047] The 200mL LB selective agar plate formulation includes: 2g tryptone, 1g yeast extract, 2g sodium chloride, 4g agar powder, 200mL ultrapure water / 50µg / mL kanamycin, 7µg / mL gentamicin, 10µg / mL tetracycline, 100µg / mL X-gal, and 40µg / mL IPTG.

[0048] 200mL LB (TC+, K+, GM+) liquid culture medium: 2g tryptone, 1g yeast extract, 2g sodium chloride, 200mL ultrapure water, 50µg / mL kanamycin, 7µg / mL gentamicin, 10µg / mL tetracycline.

[0049] 3. Expression and purification of the target product:

[0050] Sf9 cells were cultured in Sf-900 II SFM medium (Thermo Fisher: 10902-096) on a shaker at 27°C and 125 rpm. When the cells reached the logarithmic growth phase (7 × 10⁶ cells / year), the culture time was determined.6 / mL~8×10 6 At / mL, one day before transfection, the cells were diluted to 1×10⁻⁶ in Sf-900 II SFM medium. 6 3 mL was inoculated into 6-well plates. On the day of transfection, 10 μL / well of ExpiFectamine SfTm transfection reagent (Thermo Fisher: A38915) was added to 250 μL / well of Opti-MEM™ I serum-depleted medium (Gibco: 31985070) for dilution, and the mixture was inverted and incubated at room temperature for 5 min. Then, 1 μg / well of the HSV-2 gC2-gD2-gE2 recombinant Bacmid plasmid was added to the diluted transfection reagent, and the mixture was inverted and incubated at room temperature for 5 min. Finally, the above mixture was added to Sf9 cells and cultured at 27°C for 72-96 h until the Sf9 cells showed obvious pathological changes such as rounding, vacuolation, and budding. At this time, the cell supernatant contained a large amount of recombinant baculovirus containing the HSV-2 gC2-gD2-gE2 gene sequence. After centrifugation at 500 g / min for 5 min at 4°C, the supernatant was collected and recorded as the P1 generation virus stock solution. The P1 generation recombinant baculovirus was then passaged in Sf9 cells until the P3 generation. Protein expression was detected using Western blot.

[0051] Protein expression was performed on 4L Sf9 cells infected with P3 generation virus. Sf9 cells were cultured in Sf-900 II SFM medium for 3 days and then harvested for subsequent purification. Protein expression was detected using Western blot. After centrifugation, the supernatant from the harvested cell samples was collected and incubated with Ni-NTA column material to obtain the target protein. The eluted samples from the first-step purification were combined for a second-step Superdex 200 purification. The high-purity eluted samples were then concentrated and sterilely filtered through a 0.22 μm filter membrane. The final protein purity was analyzed using SDS-PAGE and Western blot. The primary antibody used for Western blot was Mouse-anti-His mAb (Proteintech: 66005-1-Ig). Protein concentration was detected using the Bradford method (with BSA as a control). HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein with a purity of over 90% was obtained, dissolved in protein storage buffer, and stored at -80°C.

[0052] Protein storage buffer formulation: 50mM Tris-HCl, 500mM NaCl, 5% (v / v) glycerol, pH 8.0.

[0053] 4. Immunogenicity evaluation of HSV-2 virus vaccine:

[0054] To test the immunogenicity of the vaccine, Balb / C mice were immunized with a mixture of the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein obtained in step 3 above and an adjuvant. All immunized Balb / C mice were purchased from the National Medical Primate Research Center; all were female, 6-8 weeks old, and weighed 18-25g. The mouse immunization experimental groups were set as follows: CpG / Alum group; gC2, gD2, gE2 group; and gC2-gD2-gE2 group. Among them, the CpG / Alum group served as a negative control for background control of the CpG / Alum adjuvant; the gC2, gD2, gE2 group served as a positive control for comparison of immunogenicity with the gC2-gD2-gE2 group of this invention. The immunization dose per mouse in the gC2-gD2-gE2 group of this invention was: 15 μg of HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein tandem antigen, 50 μg of CpG, and 125 μg of Alum. The immunization dose per mouse in the positive control group (gC2, gD2, gE2 groups) was: 5 μg of HSV-2 gC2 protein, 5 μg of HSV-2 gD2 protein, 5 μg of HSV-2 gE2 protein, 50 μg of CpG, and 125 μg of Alum. The immunization dose per mouse in the negative control group (CpG / Alum group) was: 50 μg of CpG and 125 μg of Alum. Eight mice were in each group, and all mice were immunized three times via intramuscular injection in the posterolateral aspect of the thigh on days 0, 14, and 28. On day 42, mice were euthanized by cervical dislocation, and serum and spleen lymphocytes were collected for immunogenicity evaluation. The experimental steps and results are analyzed below.

[0055] (1) Detection of specific antibody IgG:

[0056] To determine the level of specific IgG antibodies in mouse serum after three doses of vaccine, we used an enzyme-linked immunosorbent assay (ELISA) to detect the levels of specific IgG antibodies against gC2, gD2, and gE2 proteins in mouse serum.

[0057] The purified antigens gC2, gD2, and gE2 were diluted to 1 μg / mL with ELISA coating buffer (Solepro: C1050). 100 μL of the diluted protein solution was added to each well of the coated plate and incubated overnight at 4°C. The next day, the coating buffer was discarded, the plates were washed twice with PBST, and the contents were patted dry. 200 μL of PBS containing 2% BSA (goat serum) (i.e., 2% BSA, obtained by dissolving 10 g of BSA powder in 500 mL of PBS) was added to each well, and the plates were blocked at 37°C for 2 hours. During the blocking interval, mouse serum was diluted with 2% BSA (goat serum) in 96-well plates, starting with a 1:500 dilution and then serially diluted 3-fold. After blocking, the blocking buffer was discarded, the plates were washed twice with PBST, and the contents were patted dry. 100 μL of the diluted mouse serum from the 96-well plates was added to each well of the blocked plate, and the plates were incubated at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, and add 100 μL of HRP-conjugated goat anti-mouse secondary antibody (Abcam: ab6789) diluted 1:100,000 with 2% BSA (goat serum) PBS. Incubate at 37°C for 1 h. After incubation, wash four times with PBST, pat dry, add 100 μL of TMB two-component chromogenic solution (Solepro: PR12102*50), react for 5-10 min, and stop the chromogenic reaction with 50 μL of stop solution (2M H2SO4) (Solepro: C1058). Read the OD450 values ​​using a microplate reader. The smallest OD value greater than or equal to 2.1 times the OD value of the blank control well is used as the maximum dilution factor detectable by this serum. Results are as follows. Figure 1 As shown, the gC2-gD2-gE2 group of the present invention induced mice to produce high levels of specific antibody IgG. Although the level of specific IgG antibody against gC2 protein in the gC2-gD2-gE2 group was lower than that in the positive control groups gC2, gD2, and gE2, the level of specific IgG antibody against gD2 protein was higher than that in the positive control group, while the level of specific IgG antibody against gE2 protein was comparable to that in the positive control group. These results suggest that after structural upgrades and tandem fusion of the protein in the gC2-gD2-gE2 group of the present invention, the level of specific IgG antibody was not reduced compared to the existing positive control groups gC2, gD2, and gE2, but rather maintained the same antibody level.

[0058] (2) Detection of specific antibody IgG subtypes IgG1 and IgG2a:

[0059] To evaluate the IgG1 and IgG2a immune responses induced in mice by the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein vaccine, we used an enzyme-linked immunosorbent assay (ELISA) to detect the levels of specific IgG1 and IgG2a antibodies in the serum of mice after three immunizations. The experimental steps are as follows:

[0060] The purified antigens gC2, gD2, and gE2 were diluted with ELISA coating buffer (Solepro: C1050) to form a mixed antigen protein solution with a concentration of 1 μg / mL for each antigen. 100 μL of the diluted mixed antigen protein solution was added to each well of the coated plate and incubated overnight at 4°C. The next day, the coating buffer was discarded, the plate was washed twice with PBST, and the surface was patted dry. 200 μL of PBS with a BSA (goat serum) concentration of 2% was added to each well, and the plate was blocked at 37°C for 2 hours. During the blocking interval, mouse serum was diluted with PBS with a BSA (goat serum) concentration of 2% in 96-well plates, starting with a 1:500 dilution and then serially diluted 3-fold. After blocking, the blocking buffer was discarded, the plate was washed twice with PBST, and the surface was patted dry. 100 μL of the diluted mouse serum from the 96-well plate was added to the corresponding well of the blocked plate, and the plate was incubated at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, and dilute HRP-conjugated goat anti-mouse secondary antibodies IgG1 (abmam: ab97240) and IgG2a (abcam: ab97245) separately with 2% BSA (goat serum) PBS at a ratio of 1:100000. Detect these antibodies separately, meaning IgG1 and IgG2a in serum are measured in two separate plates. Add 100 μL of the diluted secondary antibody to each well and incubate at 37°C for 1 hour. After incubation, wash four times with PBST, pat dry, and add 100 μL of TMB two-component chromogenic buffer (Solepro: PR12102*50). Incubate for 5-10 minutes, and stop the chromogenic reaction with 50 μL of stop solution (2M H2SO4) (Solepro: C1058). Read the OD450 value using a microplate reader. The minimum OD value that is greater than or equal to 2.1 times the OD value of the blank control well is used as the maximum dilution factor that can be detected by the serum.

[0061] The results are as follows Figure 2 As shown, the overall serum IgG2a response level in all mice was higher than that of IgG1, but the difference was not significant. Furthermore, compared to the positive control groups gC2, gD2, and gE2, the highest dilutions of IgG1 and IgG2a in the gC2-gD2-gE2 group of this invention were at similar levels, indicating that the Th1 and Th2 immune responses induced by both groups were nearly identical, and both tended to promote a relatively balanced Th1 and Th2 response, i.e., tended to induce a relatively balanced humoral and cellular immune response. Balancing these two immune responses can effectively enhance the immunogenicity of the vaccine.

[0062] (3) Determination of neutralizing antibody titer in mouse serum:

[0063] Since HSV-1 and HSV-2 belong to the same family of herpes simplex virus, they have high homology at the gene level and in amino acid sequence, and have almost the same infection mode and latency mechanism in the human body. Therefore, neutralizing antibody tests were performed on HSV-1 and HSV-2 viruses respectively to evaluate the neutralizing ability of the HSV-2 triantigen tandem recombinant protein vaccine of the present invention against HSV-1 strain 17 (Strain17) and HSV-2 strain G (Strain G).

[0064] Mouse serum collected and frozen two weeks after three doses of vaccine were thawed at room temperature and then inactivated by incubation at 56°C for 30 min. The serum was then diluted in 96-well plates with DMEM medium (Vivacell: C3113-0500) at an initial dilution of 1:20, followed by 2-fold serial dilutions for a total of eight dilutions. Each sample was run in two replicates. Similarly, HSV-1 and HSV-2 viruses were diluted to 100 TCID50 per 50 μL using DMEM medium. The diluted serum and virus were then mixed at a 1:1 volume ratio in 96-well plates. A back-tipping experiment was also performed, with the virus solution (without serum at 100 TCID50 per 50 μL) diluted 1:10, 1:100, and 1:1000, with eight replicates for each dilution. Incubate at 37℃ for 1 hour; during the waiting period, wash Vero cells with a confluence of over 90% that have been pre-passaged twice with PBS; then, digest with 0.25% trypsin for 2 minutes, and immediately add DMEM medium containing 4% FBS (Vivacell: C04001-050) and 2% penicillin-streptomycin to terminate the digestion and resuspend the cells. After cell counting, adjust the cell suspension to 4 × 10⁶ cells / mL. 5 Cells / mL were added at 100 μL / well to the serum-virus mixture after incubation at 37°C for 1 h, and cultured in a cell culture incubator at 37°C and 5% CO2. Pathogenesis was observed after 6 days. The serum dilution corresponding to 50% cytopathic effect was defined as the neutralizing titer of the serum. The DMEM medium containing 4% FBS (Vivacell: C04001-050) and 2% penicillin-streptomycin was prepared by adding 20 mL of FBS and 10 mL of penicillin-streptomycin to 470 mL of DMEM.

[0065] The results are as follows Figure 3As shown, the neutralizing titers of the gC2-gD2-gE2 group against HSV-1 strain 17 and HSV-2 strain G were significantly higher than those of the CpG / Alum group. Furthermore, the gC2-gD2-gE2 group of this invention exhibited a neutralizing capacity comparable to that of the gC2, gD2, and gE2 groups against HSV-2 strain G. In addition, in the cross-neutralization experiment against HSV-1 strain 17, the gC2-gD2-gE2 group of this invention also showed a cross-neutralizing capacity comparable to that of the gC2, gD2, and gE2 groups. This indicates that after structural upgrades and tandem expression of the proteins in the gC2, gD2, and gE2 groups, their neutralizing capacity was not reduced due to the coverage of some antigenic epitopes after protein tandem fusion, but rather remained at the same level as before. This optimization is necessary, as it greatly improves the efficiency of vaccine preparation in the production process, saves costs, and reduces safety risks during production.

[0066] (4) Detection of cellular immune response:

[0067] To evaluate the level of specific T cell response induced by the vaccine of this invention, we used an enzyme-linked immunospot (ELISPOT) assay to detect the number of specific T cells secreting IFN-γ and IL-4 cytokines per million spleen lymphocytes, and thus judged the specific T cell response by the number of positive cells.

[0068] Two weeks after immunization with three doses of vaccine, mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol solution for 30 seconds, and their spleens were collected under aseptic conditions. A 70µm pore size cell sieve was placed in agar plates, and 4 mL of serum-free RPMI 1640 medium (Vivacell: 2441385) was added. The spleen was ground using a syringe plunger, and cells were filtered through the sieve until only white connective tissue remained. Single spleen cells were transferred to 15 mL centrifuge tubes. The tubes were centrifuged at 500g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended. 2 mL of erythrocyte lysis buffer (Solepro: R1010) was added, and the reaction was allowed to proceed for 4 minutes. Then, 10 mL of RPMI 1640 medium containing 10% FBS (50 mL of FBS was added to 450 mL of RPMI 1640 medium) was added to terminate the reaction. The tubes were centrifuged at 500g for 10 minutes at 4°C, the supernatant was discarded, and the pellet was resuspended. Add 1 mL of RPMI 1640 medium containing 10% FBS and mix thoroughly. Then perform cell counting and adjust the cell concentration to 1 × 10⁻⁶. 7cells / mL. According to the instructions of Mabtech's ELISpot Plus: Mouse IFN-γ (ALP) (Mabtech: 3321-4AST-2) and ELISpot Plus: Mouse IL-4 (ALP) (Mabtech: 3311-4APW-2) kits, prepare 250,000 pre-prepared spleen cells per well at a volume of 100 μL. Mix the pre-prepared cells with 100 μL of a mixed antigen protein stimulant (containing gC2, gD2, and gE2 proteins at a concentration of 4 μg / mL) in a 96-well plate at a 1:1 ratio. Then, slowly pipette 200 μL of the cell suspension into the wells along the sidewall of the ELISpot plate. For each sample, make two replicate wells with the stimulant and one blank background well without the stimulant. The positive control wells are stimulated with 1×PMA (Lianke Biotechnology: CS1001). Then, place the plate in a cell culture incubator at 37°C and 5% CO2 for 36 h. During this process, ensure aseptic conditions and do not move the plate. Then, following the kit instructions, the plates were washed and incubated with antibodies. After staining and air-drying the plates in the dark, cell counting was performed using a fluorescent ELISA speckle analyzer. Background interference was removed by subtracting the number of spots in the blank background wells from the number of spots in the wells with added stimulus. The obtained data were then summarized, normalized, and analyzed to calculate the number of T cells specifically secreting IFN-γ and IL-4 per million spleen cells. The RPMI 1640 medium containing 10% FBS was prepared by adding 50 mL of RPMI 1640 medium to 450 mL of FBS.

[0069] The results are as follows Figure 4 As shown, compared with the CpG / Alum group, the gC2-gD2-gE2 group of the present invention and the positive control group gC2, gD2, and gE2 groups all induced higher levels of IFN-γ+ specific T cell responses. Regarding specific IL-4+ specific T cell responses, the positive control group gC2, gD2, and gE2 groups did not induce this specific T cell, but the gC2-gD2-gE2 group of the present invention induced this specific T cell response with significant differences. This indicates that the vaccine of the present invention has advantages in inducing IL-4+ specific T cells.

[0070] (5) Detection of secreted cytokines:

[0071] To evaluate the ability of mice immunized with the gC2-gD2-gE2 vaccine to respond to Th1 and Th2 immune responses, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of Th1 cytokines IFN-γ and IL-2 and Th2 cytokines IL-4 and IL-10 in the culture supernatant of spleen lymphocytes.

[0072] The previously prepared peripheral spleen lymphocytes were seeded into 24-well cell culture plates, 1 mL of 1×10⁶ cells per well. 7 Cells were cultured in 24-well plates, each containing 100 μL of a mixed antigen protein stimulant (gC2, gD2, and gE2 proteins at a concentration of 20 μg / mL) to stimulate the cells. A positive control well was also included, containing only the positive stimulant concanavalin A (Solepro: IC4870-25 mg) at a working concentration of 10 μg / mL. All stimulants were added to the concanavalin A wells and cultured at 37°C in a 5% CO2 incubator for 48 h. The supernatant was then collected by centrifugation at 1000 rpm for 10 min at 4°C. The concentrations of cytokines IFN-γ, IL-2, IL-4, and IL-10 in the spleen lymphocyte culture supernatant were detected using the Thermo Fisher Mouse Th1 Th2 Uncoated ELISA kit (Thermo Fisher: 88-7711-44).

[0073] The results are as follows Figure 5 As shown, the gC2-gD2-gE2 group of the present invention induces mouse splenic lymphocytes to produce higher concentrations of IFN-γ and IL-2 cytokines, which are lower than those of the gC2, gD2, and gE2 groups. Although the gC2-gD2-gE2 group of the present invention shows a reduced response in Th1, it does not show a significant difference from the gC2-gD2-gE2 group. For IL-4 and IL-10, the two groups are at the same level.

[0074] Based on the above experimental data, the results indicate that the antigen tandem fusion of the gC2-gD2-gE2 group with the gC2, gD2, and gE2 groups did not decrease its immunogenicity. In the neutralization experiments on HSV-1 and HSV-2, it exhibited the same neutralizing capacity as the gC2, gD2, and gE2 groups. This optimization is extremely valuable, effectively reducing the production cost of the antigen and simplifying the process while also reducing some unknown risks during production. This invention is expected to be applied in practice, providing a new possibility for developing more efficient HSV-2 vaccines.

[0075] 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 HSV-2 tri-antigenic tandem recombinant protein vaccine composition, characterized in that, The invention includes an antigen and a complex adjuvant, wherein the antigen is a recombinant HSV-2 gC2-gD2-gE2 triantigen tandem protein, and the complex adjuvant is a CpG oligonucleotide and an aluminum adjuvant; wherein the amino acid sequence of the recombinant HSV-2 gC2-gD2-gE2 triantigen tandem protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The HSV-2 triantigenic tandem recombinant protein vaccine composition according to claim 1, characterized in that, The mass ratio of HSV-2gC2-gD2-gE2 triantigen tandem recombinant protein, CpG oligonucleotide, and aluminum adjuvant is 15:50:

125.

3. A method for preparing a HSV-2 triantigenic tandem recombinant protein vaccine, using the HSV-2 triantigenic tandem recombinant protein vaccine composition according to claim 1 or 2, characterized in that, Includes the following steps: After thawing the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein solution dissolved in protein storage buffer on ice, mix the HSV-2 gC2-gD2-gE2 triantigen tandem recombinant protein with aluminum adjuvant, and let it stand at 4°C with gentle shaking overnight. Before vaccination, mix it with CpG oligonucleotides to obtain the HSV-2 triantigen tandem recombinant protein vaccine.

4. The method for preparing the HSV-2 triantigen tandem recombinant protein vaccine according to claim 3, characterized in that, The protein storage buffer consists of 50 mM Tris-HCl, 500 mM NaCl, 5% glycerol, and pH 8.

0.

5. The method for preparing the HSV-2 triantigen tandem recombinant protein vaccine according to claim 3, characterized in that, When rocking overnight, the speed is 80 rpm.

6. The HSV-2 triantigen tandem recombinant protein vaccine prepared by the method of claim 3.

7. The use of the HSV-2 triantigen tandem recombinant protein vaccine composition according to claim 1 or 2 in the preparation of a drug for the prevention of HSV-2.

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