A herpes simplex virus type II mRNA vaccine and its application

Abstract

The present invention discloses a herpes simplex virus type II mRNA vaccine and its application, and relates to the technical field of biopharmaceuticals. The nucleic acid sequence of herpes simplex virus type II mRNA of the herpes simplex virus type II mRNA vaccine is shown in SEQ ID NO. 1. The mRNA vaccine is obtained by gene synthesis, in vitro transcription, and lipid nanoparticle encapsulation, which can quickly respond to the currently popular HSV-2 and reduce the research and development and production cycle of new vaccine development.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a herpes simplex virus type II mRNA vaccine and an application thereof. Background Art

[0002] Herpes simplex virus 2 (HSV-2) infection is a highly prevalent infectious disease that causes most cases of genital ulcers worldwide and has a significant impact on neonatal mortality. After infection, HSV-2 can be repeatedly activated, with approximately 60% of patients experiencing recurrent episodes, and 20% of patients may experience more than 10 recurrent genital ulcers within the first year after infection. In addition, HSV-2 may also be vertically transmitted during pregnancy. HSV-2 infection can increase the global incidence of HIV infection by 3 to 6 times. Therefore, the periodic recurrence of HSV-2 makes disease management more complicated, prolongs the recovery period, and seriously affects the physical and mental health of patients. Therefore, early prevention and treatment of genital herpes is clinically important for controlling the spread of HSV-2 and alleviating its adverse effects.

[0003] Currently, vaccines that have been widely studied and clinically evaluated include live attenuated vaccines, replication-deficient vaccines, DNA vaccines, subunit vaccines, and peptide vaccines. However, these vaccine candidates are still in the early stages of research or clinical trials, and although significant progress has been made in animal models, each vaccine type has its advantages and disadvantages. Therefore, there is currently no ideal HSV-2 vaccine on the market.

[0004] The natural immune system is the host's first line of defense against pathogen infection and a sentinel for detecting pathogen invasion. The host prevents HSV-2 infection through the natural immune system, while HSV-2 has evolved a variety of mechanisms to evade the corresponding antiviral response. These mechanisms involve a variety of viral proteins, especially early proteins, envelope proteins, etc., which can target the host's antiviral signaling pathways. The protein encoded by UL41 is a ribonuclease, also known as viral host shutoff protein (VHS), which can degrade host mRNA, such as cGAS mRNA, and inhibit the cGAS / STING-mediated DNA recognition pathway; it can also target the host cell restriction factor zinc finger antiviral protein (ZAP) and inhibit its expression by degrading it, thereby achieving immune escape.

[0005] To further confirm the role of UL41 mRNA vaccine in HSV-2 infection, the immunogenicity of HSV-2 UL41 mRNA vaccine will be elucidated, the effect of HSV-2 UL41 mRNA in preventing HSV-2 infection will be evaluated, and the role of UL41 mRNA vaccine in the immune mechanism against HSV-2 infection will be clarified, providing a new theoretical basis for the prevention and treatment of recurrent herpes caused by HSV-2. Summary of the invention

[0006] The purpose of the present invention is to provide a herpes simplex virus type II mRNA vaccine and its application. The mRNA vaccine has a short development time and high safety, can produce immune protection against the current HSV-2 prevention and control, and provide technical support for HSV-2 prevention and control.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A herpes simplex virus type II mRNA vaccine, wherein the nucleic acid sequence of the herpes simplex virus type II mRNA of the herpes simplex virus type II mRNA vaccine is shown in SEQ ID NO. 1.

[0009] Furthermore: the amino acid sequence of the viral protein UL41 of the herpes simplex virus type II mRNA is shown in SEQ ID NO. 2.

[0010] Further: The method for preparing the herpes simplex virus type II mRNA vaccine comprises:

[0011] The HSV-2 viral protein UL41 DNA sequence is synthesized, the DNA sequence is constructed on a vector, a linearized DNA template is prepared, in vitro transcription is performed, the DNA template is removed, the in vitro transcribed mRNA product is purified and quality tested, and then mixed with a lipid nanoparticle delivery system.

[0012] Further: The lipid nanoparticle preparation method comprises:

[0013] Step 1) preparing an organic phase: dissolving cationic liposome SM102, cholesterol, DSPC, and PEG-DMG in anhydrous ethanol to obtain an organic phase for standby use;

[0014] Step 2) preparing an aqueous phase: dissolving the mRNA in a citrate buffer to obtain an aqueous phase for later use;

[0015] Step 3) Preparation of lipid nanoparticles: The organic phase and the aqueous phase are mixed to obtain a mixed solution, the mixed solution is diluted with a PBS solution, the ethanol in the mixed solution is removed by an ultrafiltration tube, and the solution is concentrated to obtain lipid nanoparticles for encapsulating mRNA.

[0016] Further: the removal of the DNA template is performed after the pre-sequence reaction is completed, DNase I is added to the transcription product and mixed evenly, centrifuged instantly to make the solution all gather at the bottom of the tube, and then placed on a thermal cycler for incubation.

[0017] Further: the in vitro transcribed mRNA product purification reaction system includes: RNA transcription product after template removal, lithium chloride (LiCl), RNase free water (RNase free H2O), the purification reaction system is mixed and then divided into two tubes for precipitation;

[0018] After precipitation, centrifuge at low temperature to precipitate RNA;

[0019] After discarding the supernatant, wash the precipitate with ethanol and centrifuge again;

[0020] Discard the supernatant, air-dry at room temperature, dissolve the RNA in the solution, take a portion for quantification, and aliquot the rest for storage.

[0021] Furthermore: the HSV-2 UL41 mRNA transfection cells of the herpes simplex virus type II mRNA vaccine include: cell recovery, cell passaging, and vaccine treatment.

[0022] Further: the cell recovery is as follows: taking out the frozen cells from liquid nitrogen and shaking them in a water bath, taking them to an ultra-clean workbench after they are dissolved, transferring the cells to a centrifuge tube containing complete culture medium, and mixing them evenly;

[0023] Discard the supernatant, add DMEM complete medium, mix by pipetting, transfer the cell suspension into a culture flask, and culture in an incubator.

[0024] Further: the cell passage is as follows: on a clean bench, the culture medium in the cell culture dish is aspirated and sterile PBS is added from the edge of the culture dish;

[0025] Trypsin was added for digestion. When most of the cells became round and separated from each other, DMEM complete medium containing fetal bovine serum was added to terminate the digestion. Single-cell suspension was made by pipetting and centrifugation. The medium was discarded and DMEM complete medium containing fetal bovine serum was added again to resuspend the cells. The cells were then aliquoted into culture bottles for continued culture.

[0026] Further: the vaccine treatment is: diluting LNP-(HSV-2 UL41 mRNA + Luci) to 10 mg / ml with culture medium, adding it to cells, treating it for 24-48 hours, and observing the fluorescence.

[0027] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0028] (1) The mRNA vaccine obtained through gene synthesis, in vitro transcription, and lipid nanoparticle encapsulation can quickly respond to the currently prevalent HSV-2 and reduce the R&D and production cycle of new vaccine development.

[0029] (2) The RNA vaccine prepared by the present invention has high production efficiency, can produce a high level of cellular immune response in the body and reduce inflammatory response, can effectively reduce the incidence of HSV-2 infection in mice, has great promotion value for the prevention and control of HSV-2 virus, and provides an important means for the prevention and control of HSV-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the result of double enzyme digestion identification of the recombinant plasmid V0301-UL41 and linearization of the in vitro transcription template DNA in the present invention; wherein, Figure 1 In A, lane 1: V0301 plasmid; lane 2: agarose gel electrophoresis of the recombinant plasmid V0301-UL41 digestion identification; Figure 1 In B, lane 1: linearization result of in vitro transcription template DNA. Marker: KB ladder 10000.

[0031] Figure 2 This is a gel retardation gel map of the present invention; wherein, lane 1: HSV-2 UL41 mRNA complex 1 mg, lane 2: HSV-2 UL41 mRNA complex 2 mg, lane 3: LNP-UL41 mRNA complex 2 mg + RNase.

[0032] Figure 3 The results of the HSV-2 UL41 mRNA complex transfection efficiency and CCK8 toxicity test in the present invention; wherein, Figure 3 A is the fluorescence observation of 293T cells transfected with LNP-(UL41+Luci) vaccine. Figure 3 B is the result of cytotoxicity test of LNP-(UL41+Luci) on 293T cells.

[0033] Figure 4 The weight and survival rate of mice immunized with the HSV-2 UL41 mRNA vaccine of the present invention; wherein, Figure 4 A shows the weight changes of mice in the normal group, wild virus group and HSV-2 UL41 mRNA vaccine muscle immunization group within 60 days; Figure 4 B shows the changes in mouse survival rate within 60 days in the normal group, wild virus group and HSV-2 UL41 mRNA vaccine muscle immunization group.

[0034] Figure 5 CD4 + T, CD8 + T cell, Th1, Th2 cell flow cytometry results; among them, Figure 5 A is the control group CD4 + flow cytometry results of T cells; Figure 5 B is CD4 in muscle immune group + Flow cytometry results of T cells; Figure 5 C is the CD4 of the control group and muscle immunization group + T cell statistical analysis chart; Figure 5 D is the control group CD8 + Flow cytometry results of T cells; Figure 5 E is CD8 in muscle immunization group + Flow cytometry results of T cells; Figure 5 F is the CD8 in the control group and muscle immunization group + T cell statistical analysis chart; Figure 5 G is the flow cytometry results of Th1 cells in the control group; Figure 5 H is the flow cytometry result of Th1 cells in the muscle immunization group; Figure 5 I is the statistical analysis diagram of Th1 cells in the control group and muscle immunization group; Figure 5 J is the flow cytometry result of Th2 cells in the control group; Figure 5 K is the flow cytometry result of Th2 cells in the muscle immunization group; Figure 5 L is the statistical analysis chart of Th2 cells in the control group and muscle immunization group.

[0035] Figure 6 is the body weight and survival rate of the normal group, immune group and wild-type group in the present invention; wherein, Figure 6 A is the weight change of mice in the HSV-2 UL41mRNA vaccine muscle immunization group, wild virus group and normal group within 10 days after the virus challenge; Figure 6 B shows the changes in mouse survival rate within 10 days after infection in the HSV-2UL41 mRNA vaccine muscle immunization group, wild virus group and normal group.

[0036] Figure 7 The vaginal tissue and spleen pathological results and the vaginal tissue inflammatory factor secretion results of the normal group, immune group and wild-type group in the present invention; wherein, Figure 7 A is the vaginal tissue pathological results of the normal group, immune group and wild-type group; Figure 7 B is the spleen tissue pathological results of the normal group, immune group and wild-type group; Figure 7 C is the results of IL-6 secretion in vaginal tissue of normal group, immune group and wild-type group; Figure 7 D is the results of TNF-a secretion in vaginal tissue of normal group, immune group and wild-type group; Figure 7 E is the results of IFN-γ secretion in vaginal tissue of normal group, immune group and wild-type virus group. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Embodiment 1:

[0039] The HSV-2 viral protein UL41 DNA sequence is synthesized, the DNA sequence is constructed on a vector, a linearized DNA template is prepared, in vitro transcription is performed, the DNA template is removed, the in vitro transcribed mRNA product is purified and quality inspected, and then mixed with a lipid nanoparticle (LNP) delivery system to make an mRNA vaccine. This mRNA vaccine is the herpes simplex virus type II mRNA vaccine, and the herpes simplex virus type II mRNA vaccine is abbreviated as HSV-2 UL41 mRNA.

[0040] The kit used for in vitro transcription of mRNA is Thermo's T7 High Yield Transcription Kit. Use a pipette to gently mix the reaction system (do not shake it violently), centrifuge it instantly to ensure that all the solution is collected at the bottom of the tube, and place it in a thermal cycler to incubate the reaction at 37°C for 2-4 hours (the reaction time can be adjusted and optimized appropriately according to the length of the synthesized RNA). After the reaction, place the system on ice.

[0041] To remove the DNA template, after the pre-sequence reaction is completed, add 40 ml DNase I (1U / ml) to the transcription product and mix evenly with a pipette. Centrifuge instantly to allow all the solution to pool at the bottom of the tube, and incubate the reaction at 37°C in a thermal cycler for 30 minutes to completely eliminate the DNA template in the system.

[0042] Purification of in vitro transcribed mRNA products is to prepare the reaction system, remove the template RNA transcription product 400ul, LiCl (lithium chloride) 400ml, RNase free H2O (RNase-free water) and increase the reaction system volume to 1000ul, gently mix to make it fully mixed, then divide into two tubes and precipitate overnight at -20℃. After precipitation, use a low-temperature refrigerated centrifuge to centrifuge at 4℃ 13000rpm for 30min to precipitate RNA. After discarding the supernatant, wash the precipitate with 1mL of ethanol, centrifuge again, and repeat twice. Discard the supernatant, air dry at room temperature, add 1ml solution to dissolve the RNA, take part of the quantitative, and store the rest at -80℃ after packaging.

[0043] The purity of mRNA was determined by using the Agilent RNA 6000 Nano kit #5067-1511 kit, which was equilibrated to room temperature in advance. 65 ml of gel, 1 ml of fluorescent dye was added, mixed thoroughly, and centrifuged at 14,000 rpm for 10 min at RT. The ladder (invitrogene AM7150) was removed, denatured at 70°C for 2 min, and placed on ice for later use. The chip was removed, 9 ml of gel complex was added to the G well, and the gel was pressed for 30 s (using the matching equipment); after 30 s, the fixed lock was opened, and the propeller was slowly bounced up to about 0.7 ml, and the propeller was gently raised to 1 ml manually. 9 ml of gel was added to each of the other two wells. 5 ml of internal standard was added to all test wells, 1 ml of ladder was added to the lower right well, and 1 ml of mRNA, about 200 ng, was added to all test wells. The matching shaker had a maximum speed of 2 min. Bioanalizer test: Open the 2100 export software and run the program. Take a small amount of RNA product and use a micro-spectrophotometer (NAnodrop) or QUBIT to detect the concentration and purity of RNA. The test data are shown in Table 1:

[0044] Table 1 HSV-2 mRNA product test results

[0045]

[0046] Among them, the mRNA vaccine encapsulated by lipid nanoparticles, when preparing lipid nanoparticles, cationic liposome SM102, cholesterol, DSPC and PEG-DMG are dissolved in anhydrous ethanol, and after being dissolved in anhydrous ethanol, cationic liposome SM102, cholesterol, DSPC and PEG-DMG are dissolved in anhydrous ethanol at a mass molar ratio of 50:38.5:10:1.5 to obtain an organic phase; when preparing lipid nanoparticles, mRNA is dissolved in citrate buffer and the concentration is adjusted to 0.1 mg / ml to obtain an aqueous phase. The organic phase and the aqueous phase are mixed using a microfluidic device, the volume ratio of the organic phase to the aqueous phase is 1:3, and the flow rate during mixing is 2 ml / min; the mixed solution is immediately diluted 50 times with PBS solution (phosphate buffer solution), and the ethanol component in the solution is removed using a 300kDa ultrafiltration tube, and the ethanol component in the solution is removed three times, and the final solution is concentrated to an mRNA concentration of 0.2 mg / ml to obtain lipid nanoparticles encapsulating mRNA.

[0047]

[0048] The amino acid sequence of viral protein UL41 is shown in SEQ ID NO. 2; specifically, it is as follows: MGLFGMMKFAQTHHLVKRRGLRAPEGYFTPIAVDLWNVMYTLVVKYQRRYPSYDREAITLHCLCSMLRVFTQKSLFPIFVTDRGVECTEPVVFGAKAILARTTAQCRTDEEASDVDASPPPSPITDSRPSFAFSNMRRRGHAFAPGDRGTRAAGPGPAAPSGAPSKPALRLAHLFCIRVLRALGYAYINSGQLEADDACANLYHTNTVAYVHTTDTDLLLMGCDIVLDISTGYIPTIHCRDLLQYF KMSYPQFLALFVRCHTDLHPNNTYASVEDVLRECHWTAPSRSQARRAARRERANSRSLESMPTLTAAPVGLETRISWTEILAQQIAGEDDYEEDPPLQPPDVAGGPRDGARSSSSEILTPPEL VQVPNAQRVAEHRGYVAGRRRHVIHDAPEALDWLPDPMTIAELVEHRYVKYVISLISPKERGPWTLLKRLPIYQDLRDEDLARSIVTRHITAPDIADRFLAQLWAHAPPPAFYKDVLAKFWDE.

[0049] Embodiment 2:

[0050] After the herpes simplex virus type II mRNA vaccine is constructed, the particle size and encapsulation efficiency are tested.

[0051] The particle size was detected by a particle size analyzer. The encapsulation rate was detected using the Invitorgen kit Quant-iTRiboGreen RNA Reagent and Kit. The UL41 gene fragment was amplified using the HSV-2 viral genome as a template, and after double digestion with EcoRI and HindIII, it was connected to the double-digested V0301 vector for double digestion verification. The obtained DNA template was used for in vitro transcription of mRNA using the T7 High Yield Transcription Kit and the mRNA was purified using the RNA 6000Nano Kit. The purity of HSV-2 UL41 mRNA reached more than 90%, the capping rate reached more than 95%, and the quality inspection was qualified. The particle size reached 82.5, and the quality inspection was qualified.

[0052] Double restriction enzyme digestion identification of recombinant plasmid V0301-UL41 and linearization results of in vitro transcription template DNA, such as Figure 1 As shown in A, two fragments appeared in the electrophoresis, about 4100 bp and 1700 bp, and the size of the enzyme-cut fragment was consistent with the expected value. After the vector construction was completed, the plasmid was linearized and detected by 1% agarose gel electrophoresis. Figure 1 B The result shows that the size of the vector linearization band is consistent with the expectation.

[0053] The prepared HSV-2 UL41 mRNA encapsulation products were tested for encapsulation efficiency, particle size, nanoparticle dispersion coefficient, and gel retardation. Figure 2 As shown, the results show that the encapsulation rate reaches more than 90%, with high encapsulation efficiency and good encapsulation effect. Table 2 shows the particle size and encapsulation rate test results of HSV-2 mRNA products.

[0054] Table 2 HSV-2 mRNA product particle size and encapsulation efficiency test results

[0055]

[0056] Embodiment 3:

[0057] HSV-2 UL41 mRNA transfection of cells includes the following steps:

[0058] Cell recovery: When the water bath temperature rises to 37°C, take out the frozen cells from the liquid nitrogen according to the record, shake them quickly in the 37°C water bath, and quickly take them to the clean bench after they are dissolved. Transfer the cells to a 15ml centrifuge tube containing 10ml complete medium, mix them, and centrifuge them at room temperature for 5 minutes at 1000r / min. Pour out the supernatant, add DMEM (DMEM medium is a medium containing various amino acids and glucose) complete medium, mix them by blowing, and transfer the cell suspension into a T-25 culture bottle, and culture them in an incubator at 37°C and 5% CO2.

[0059] Cell passaging: When the cells grow to 80%, use a pipette to carefully discard the culture medium in the cell culture dish on the clean bench, slowly add 3 ml of sterile PBS (phosphate buffered saline) from the edge of the culture dish with a pipette, discard the PBS with a pipette, and repeat washing twice; add an appropriate amount of 0.25% trypsin for digestion, and when most of the cells become round and separate from each other, add an appropriate amount of DMEM complete medium containing fetal bovine serum to terminate digestion, gently blow to make a single cell suspension, centrifuge at 1000r / min for 5min, discard the culture medium, re-add DMEM complete medium containing fetal bovine serum to resuspend the cells, and then divide into culture bottles for continued culture, and pass them at a ratio of 1:3.

[0060] Vaccine treatment: Dilute LNP-(HSV-2 UL41 mRNA + Luci) to 10 mg / ml with culture medium, add it to cells and observe the fluorescence after 24-48 hours of treatment.

[0061] The protein encoded by UL41 is an endoribonuclease also known as virionhost shutoff protein (VHS), which can degrade host mRNA, such as cGAS mRNA to block the cGAS / STING-mediated DNA recognition signaling pathway. It can also target the host cell restriction factor zinc finger antiviral protein (ZAP) with antiviral activity and degrade it, inhibiting its expression to achieve immune escape. HSV-2 UL41mRNA complex transfection efficiency and CCK8 kit toxicity test, Luci-mRNA (mRNA expressing fluorescent protein after transfection of cells) and UL41 mRNA are simultaneously encapsulated in LNP (liposome), that is, LNP-(UL41+Luci). After treating 293T cells with 10mg of LNP-(UL41+Luci) for 24h, the fluorescence intensity of 293T cells is observed using a live imaging instrument. Figure 3 A The results show that the fluorescence of the LNP-(UL41+Luci) group is strong, and the normal group has no fluorescence, indicating that LNP-(UL41+Luci) enters the cells and its transfection efficiency is high. The CCK8 kit was used to detect the cytotoxicity of LNP-(UL41+Luci). Figure 3 As shown in B, compared with NC (control vaccine group), the addition of LNP-(UL41+Luci) had no significant effect on cell activity.

[0062] Embodiment 4:

[0063] BALB / c mice were divided into 3 groups, namely normal group, wild strain group and muscle immunization group. The immunization times were 2 times, with an interval of 4 weeks. The normal group and wild strain group were injected with normal saline, with 10 mice in each group; the muscle immunization group was injected with 10μg mRNA vaccine, with 10 mice in each group. One month after the completion of immunization in the immunized group, a lethal dose of virus was carried out at the same time. One day, four days and seven days after the virus was challenged, the acute infection of HSV-2 wild strain was completed and the samples were collected. Among them, the weight and survival of the mice were continuously observed, and the weight and survival curves were prepared. For the pathological examination of vaginal tissue and spleen tissue, the spleen tissue and the tissue of the virus site were soaked in formalin, dehydrated, transparent, paraffin-embedded, sliced, and H&E stained, and the pathological changes of the tissue were observed under the microscope. Secretion of inflammatory factors: Total RNA was extracted from vaginal and spleen tissues of mice at different time points, and the transcription levels of IFN-α, TNF-γ, and IL-6 were detected by relative quantitative PCR using the One Step TB Green® PrimeScriptTM PLUS RT-PCR Kit (Perfect Real Time).

[0064] Figure 4 The results showed that the weight of mice in the HSV-2 UL41 mRNA vaccine group was not affected, and no deaths occurred. Therefore, the HSV-2 UL41 mRNA vaccine has good safety. Figure 4 As shown in the figure, the body weight of mice in the muscle immunization group was not affected and no death occurred. After two immunizations at 0d and 28d (immunization dose of 10μg), the cellular immune response in the animals was successfully induced. Figure 5 As shown, compared with the normal group, the CD4 + T, CD8 + The proportion of T cells increased significantly, and the difference was significant, among which the proportion of Th1 cells secreting IFN-γ and Th2 cells secreting IL-4 increased significantly. Figure 6 A. Figure 6 In B, vaginal challenge was performed 4 weeks after two immunizations. The results showed that compared with the normal group, the wild strain group had a significant decrease in body weight and death, while the immunized group had a slight decrease in body weight and no death. Figure 7 A. Figure 7 B. Pathological results showed that compared with the normal group, the wild strain group had epithelial cell shedding and inflammatory cell infiltration in the vaginal tissue, hemorrhage and inflammatory cell infiltration in the spleen tissue, and the immune group had milder symptoms than the wild strain group. Figure 7 C. Figure 7 D. Figure 7E Tissues from the infected area were taken to detect inflammatory cytokines. The results showed that compared with the normal group, IL-6, TNF-, and IFN-γ in the wild strain group were significantly higher than those in the normal group and the immune group. These three inflammatory cytokines in the immune group increased on the 4th day and returned to normal levels on the 7th day.

[0065] Table 3 Statistical analysis of mouse body weight within 7 days after challenge

[0066]

[0067] Note: * represents the comparison with the normal group; # represents the comparison with the model group; * / # represents P <0.05; ** represents P <0.01; *** represents P <0.001.

[0068] One month after the immunization, the spleen lymphocytes of the normal group and the HSV-2 UL41 mRNA vaccine muscle immunization group were separated, and the spleen T lymphocytes of the mice were analyzed by flow cytometry. The scheme used was: CD3, CD4, CD8, IFN-γ and IL-4. The results showed that compared with the normal group, the HSV-2 UL41 mRNA vaccine immunization group had CD4 + T and CD8 + T cells were significantly upregulated, and Th1 and Th2 cells were also upregulated to varying degrees. Figure 7 As shown, the results showed that HSV-2 UL41 mRNA vaccine can activate cellular immunity in the spleen tissue of mice after immunization.

[0069] HSV-2 UL41 mRNA vaccine can prevent death caused by HSV-2, but cannot avoid inflammatory response at the challenge site. After two immunizations, mice were challenged with a lethal dose of wild-type HSV-2. Figure 7 It can be seen that the weight of the wild virus group and the immune group decreased, there was no death in the immune group, and the survival rate was 100%.

[0070] The weight of mice in the HSV-2 UL41 mRNA vaccine muscle immunization group, wild virus group and normal group within 10 days after the virus attack was statistically analyzed. As shown in Table 3, the weight of mice in the wild virus group decreased significantly from 1 to 5 days, while the weight of the immunized group on the 3rd and 4th days was significantly different from that of the wild virus group, and there was no significant difference in weight loss compared with the control group. The results show that the HSV-2 UL41 mRNA vaccine can protect mice from death caused by HSV-2 wild virus attack.

[0071] Pathological results showed that compared with the normal group, the wild-type group had epithelial cell shedding and inflammatory cell infiltration in the vaginal tissue, and hemorrhage and inflammatory cell infiltration in the spleen tissue. The immune group had milder symptoms than the wild-type group, such as Figure 7 A and Figure 7 B. The tissues of the infected parts were taken to detect inflammatory cytokines. The results showed that compared with the normal group, the wild strain group had significantly higher levels of IL-6, TNF-a, and IFN-γ than the normal group and the immune group. The immune group had significantly higher levels of these three inflammatory cytokines than the normal group on the 4th day, and returned to normal levels on the 7th day. Figure 7 C-7E.

[0072] It is known that HSV-2 UL41 mRNA vaccine can activate cellular immunity in mice and alleviate inflammatory response, which is related to the regulation of UL41 on the host's immune response, including innate immunity and adaptive immunity.

[0073] Although the present invention is described herein with reference to a number of illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, drawings, and claims, a variety of variations and modifications may be made to the components and / or layout of the subject combination layout. In addition to the variations and modifications made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A herpes simplex virus type II mRNA vaccine, characterized in that: The nucleic acid sequence of the herpes simplex virus type II mRNA of the herpes simplex virus type II mRNA vaccine is shown in SEQ ID NO.

1.

2. A herpes simplex virus type II mRNA vaccine according to claim 1, characterized in that: The amino acid sequence of the viral protein UL41 of the herpes simplex virus type II mRNA is shown in SEQ ID NO.

2.

3. A herpes simplex virus type II mRNA vaccine according to claim 1, characterized in that: The method for preparing the herpes simplex virus type II mRNA vaccine comprises: The HSV-2 viral protein UL41 DNA sequence was synthesized, the DNA sequence was constructed on a vector, a linearized DNA template was prepared, in vitro transcription was performed, the DNA template was removed, the in vitro transcribed mRNA product was purified and quality tested, and then mixed with a lipid nanoparticle delivery system.

4. A herpes simplex virus type II mRNA vaccine according to claim 3, characterized in that: The lipid nanoparticle preparation method comprises: Step 1) preparing an organic phase: dissolving cationic liposome SM102, cholesterol, DSPC, and PEG-DMG in anhydrous ethanol to obtain an organic phase for standby use; Step 2) preparing an aqueous phase: dissolving the mRNA in a citrate buffer to obtain an aqueous phase for later use; Step 3) Preparation of lipid nanoparticles: The organic phase and the aqueous phase are mixed to obtain a mixed solution, the mixed solution is diluted with a PBS solution, the ethanol in the mixed solution is removed by an ultrafiltration tube, and the solution is concentrated to obtain lipid nanoparticles for encapsulating mRNA.

5. A herpes simplex virus type II mRNA vaccine according to claim 3, characterized in that: The DNA template is removed by adding DNase I to the transcription product after the pre-sequence reaction is completed, mixing evenly, centrifuging instantly to make the solution all gather at the bottom of the tube, and incubating on a thermal cycler.

6. A herpes simplex virus type II mRNA vaccine according to claim 3, characterized in that: The purification reaction system of the in vitro transcribed mRNA product comprises: the RNA transcription product after the template is removed, lithium chloride, and RNase-free water; the purification reaction system is mixed and then divided into two tubes for precipitation; After precipitation, centrifuge at low temperature to precipitate RNA; After discarding the supernatant, wash the precipitate with ethanol and centrifuge again; Discard the supernatant, air-dry at room temperature, dissolve the RNA in the solution, take a portion for quantification, and aliquot the rest for storage.

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