Novel adjuvant subunit rabies virus vaccine as well as preparation method and application thereof

Abstract

The invention provides a novel adjuvant subunit rabies virus vaccine as well as a preparation method and application thereof, the vaccine takes a rabies virus (RABV) glycoprotein extracellular domain prepared by a gene recombination technology as an antigen, and takes a combination of CpG oligodeoxynucleotide and QS21 as an adjuvant. A contrast experiment is carried out on the vaccine, an LNP-mRNA-G-H270P vaccine, a commercial inactivated vaccine and an aluminum adjuvant subunit vaccine, and the result shows that the vaccine can induce a remarkably higher RABV-G specific IgG antibody and virus neutralizing antibody titer, and the humoral immune response is superior to that of other vaccines; meanwhile, moderate and effective Th1 type cell immune response can be induced, so that protective immunity can be supported; 100% protection is achieved in a lethal RABV attack experiment, and the protection effect is equivalent to that of an mRNA vaccine and is remarkably superior to that of an aluminum adjuvant vaccine and a commercial inactivated vaccine. The vaccine provided by the invention has a controllable immune activation mechanism and relatively high safety, avoids the storage risk and excessive inflammatory response of a nucleic acid technology, and is a more reliable and universal choice in the current vaccine technology.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, and specifically relates to a novel adjuvant subunit rabies virus vaccine, its preparation method, and its application. Background Technology

[0002] Rabies, a deadly zoonotic disease caused by the rabies virus (RABV), remains a major global public health challenge, causing approximately 59,000 deaths annually. Asia and Africa are particularly hard hit due to a lack of post-exposure prophylaxis (PEP) resources and weak cold chain infrastructure. Once RABV invades the central nervous system, the mortality rate is nearly 100% after the onset of clinical symptoms, making the development of highly effective and readily available preventative vaccines an urgent priority.

[0003] Current rabies vaccine technology has significant limitations:

[0004] Traditional inactivated vaccines (such as human diploid cell vaccines and Vero cell vaccines) are the gold standard for PEP, but they require multiple intramuscular injections, resulting in high production costs. Furthermore, in resource-limited areas, they are difficult to fully exert their protective effect due to insufficient cold chain conditions and fluctuations in immunogenicity.

[0005] While mRNA vaccines (such as the LNP-encapsulated H270P mutant RABV-G vaccine) have shown the advantage of rapidly inducing a strong immune response, they rely on ultra-low temperature cold chain storage and transportation at -70°C, resulting in extremely high costs. Furthermore, nucleic acid technology may trigger excessive inflammatory responses, limiting their widespread adoption in underdeveloped regions.

[0006] Subunit vaccines, based on recombinant RABV antigens (such as glycoprotein RABV-G), are characterized by high safety and controllable production, but they heavily rely on adjuvants to enhance immunogenicity. Currently widely used aluminum adjuvants can only enhance humoral immunity to a limited extent, and the production of rabies virus neutralizing antibodies (RVNA) is delayed, making it difficult to meet the need for rapid protection.

[0007] To overcome these bottlenecks, the development of novel adjuvants has become crucial for improving the performance of subunit vaccines. Toll-like receptor (TLR) agonists such as CpG and QS21 have been shown to have unique advantages: CpG can activate TLR9, driving Th1-type immune responses and IFN-γ secretion; QS21, as a saponin derivative, can potently enhance antibody responses and specific T-cell responses. Both, alone or in combination with other adjuvants, have shown potential in vaccines for coronaviruses and influenza, but in the field of rabies, how to achieve rapid and efficient humoral immunity, moderate and effective cellular immunity, and comprehensive performance without the need for extreme cold chains through the synergistic effect of adjuvants remains an unsolved technical challenge.

[0008] In existing technologies, the adjuvant selection for subunit vaccines is mostly limited to single components (such as aluminum salts) or non-synergistic combinations, which cannot balance the strength of immunity, the speed of protection and the feasibility of storage and transportation; while mRNA vaccines, although highly immunogenic, are limited by cold chain and safety risks.

[0009] Therefore, developing a novel rabies vaccine that integrates high humoral immune activity, moderate cellular immunity, convenient storage and transportation, and controllable safety has become a core requirement for overcoming existing technological bottlenecks. Summary of the Invention

[0010] In view of the problems existing in the prior art, the purpose of this invention is to improve the immunogenicity, in vivo protection and immune function such as virus clearance of RABV subunit vaccines, thereby providing a novel adjuvant subunit rabies virus vaccine, its preparation method and application.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] The first aspect of this invention provides a novel adjuvant subunit rabies virus vaccine, the vaccine comprising an antigen and an adjuvant;

[0013] The antigen is the extracellular domain of a rabies virus (RABV) glycoprotein;

[0014] The adjuvant is a combination of CpG oligodeoxynucleotide and QS21.

[0015] More preferably, the extracellular domain of the RABV glycoprotein is the extracellular segment of the recombinantly expressed RABV-G protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0016] More preferably, the mass ratio of CpG oligodeoxynucleotides to QS21 in the adjuvant is 1 to 3:1.

[0017] More preferably, the concentration of antigen in the vaccine is 5 μg / injection, the total amount of adjuvant added is 15 μg / injection, and the mass ratio of CpG oligodeoxynucleotides to QS21 is 2:1.

[0018] A second aspect of this invention provides a method for preparing the novel adjuvant subunit rabies virus vaccine described in the first aspect, comprising the following steps:

[0019] (1) The extracellular domain of RABV glycoprotein was prepared by gene recombination and purified for later use;

[0020] (2) The purified RABV glycoprotein extracellular domain is mixed with adjuvant QS21 and CpG oligodeoxynucleotide in sterile phosphate-buffered saline to obtain the subunit rabies virus vaccine.

[0021] A third aspect of the present invention provides the application of the novel adjuvant subunit rabies virus vaccine described in the first aspect, the application comprising:

[0022] 1) To prepare a pharmaceutical composition for the prevention or treatment of diseases caused by rabies virus infection;

[0023] 2) Prepare a pharmaceutical composition for the prevention or treatment of rabies virus infection.

[0024] More preferably, the pharmaceutical composition is administered via intramuscular, subcutaneous, or intradermal injection.

[0025] More preferably, the pharmaceutical composition is used to induce the body to produce specific IgG antibodies and virus-neutralizing antibodies against RABV, and to induce a Th1-type cellular immune response.

[0026] The novel adjuvant subunit rabies virus vaccine, its preparation method, and its application provided by this invention have the following significant advantages compared to existing technologies:

[0027] I. Significantly enhanced humoral immune response provides key protection for post-exposure prophylaxis.

[0028] The G+QS21+CpG subunit vaccine of this invention induces significantly higher levels of RABV-G specific IgG antibodies and virus-neutralizing antibodies (RVNA). Experimental data show that in the late immunization period (42 days after the first immunization), the vaccine induced a RABV-G specific IgG titer of 2,918,400, which is 3.08 times that of the LNP-mRNA-G-H270P vaccine, 45.6 times that of the aluminum adjuvant subunit vaccine, and 145.77 times that of the commercial inactivated vaccine; its RVNA titer is as high as 2246 IU / mL, which is 3 times that of the LNP-mRNA-G-H270P vaccine, 38 times that of the aluminum adjuvant vaccine, and 115 times that of the commercial inactivated vaccine. This potent and long-lasting humoral immune response enables rapid seroconversion, which is crucial for post-exposure prophylaxis (PEP) of rabies, effectively preventing viral invasion of the central nervous system and significantly reducing the risk of disease.

[0029] II. The cellular immune response is moderate and effective, synergistically enhancing protective efficacy.

[0030] Although the LNP-mRNA-G-H270P vaccine has advantages in cellular immunity, the G+QS21+CpG subunit vaccine of this invention can still induce a significant Th1-type cellular immune response: the levels of IFN-γ-secreting spleen cells, IL-2 levels in spleen cell supernatant, and IFN-γ-producing CD4+ T cells induced by it are significantly higher than those in the PBS control group, and are sufficient to support protective immunity. This immune response mode of "humoral immunity as the mainstay and cellular immunity as the secondarystay" can both rapidly neutralize the virus through antibodies and clear potentially infected cells through cellular immunity, forming a synergistic protective mechanism.

[0031] Third, it provides excellent in vivo protective effects, comparable to mRNA vaccines, with more controllable safety.

[0032] In a lethal rabies virus (50LD50 CVS-11 strain) challenge experiment, the G+QS21+CpG subunit vaccine achieved a 100% mouse survival rate, comparable to the protective efficacy of the LNP-mRNA-G-H270P vaccine, and significantly superior to aluminum adjuvant vaccines (70% survival rate) and commercial inactivated vaccines. Furthermore, mice immunized with this vaccine maintained stable body weight and showed no significant clinical symptoms after viral challenge, indicating that it not only prevents death but also maintains the body's health.

[0033] More importantly, its immune activation mechanism is controllable, avoiding the excessive inflammatory response that mRNA vaccines may cause, resulting in higher safety and reducing the risk of immune-related adverse reactions.

[0034] Fourth, it has lower storage and transportation costs, greater versatility, and is more suitable for areas with limited resources.

[0035] Existing mRNA vaccines (such as LNP-mRNA-G-H270P) require cold chain storage and transportation at -70°C, which is costly and has strict infrastructure requirements, making them difficult to distribute in areas with weak cold chain conditions. In contrast, the novel adjuvant subunit vaccine of this invention does not require extreme low-temperature storage, exhibits better stability, and can adapt to a wider range of storage and transportation environments. This significantly reduces the cost and difficulty of promotion, making it particularly suitable for resource-limited regions in Asia and Africa where rabies is prevalent, helping to address the high mortality rate caused by insufficient accessibility to PEP in these areas.

[0036] V. The preparation process is simple, the production cost is controllable, and it is easy to scale up production.

[0037] The subunit vaccine of this invention uses the extracellular domain of RABV-G prepared by gene recombination technology as the antigen, combined with QS21+CpG adjuvant. The preparation process does not require complicated LNP encapsulation or virus inactivation steps, making the process simpler. It can be prepared on a large scale through conventional biological agent production processes, and the production cost is lower than that of mRNA vaccines and traditional cell culture inactivated vaccines, which helps to reduce vaccination costs and increase population coverage.

[0038] VI. It has a wide range of applications, flexible administration methods, and is easy to promote in clinical practice.

[0039] This vaccine can be administered via conventional routes of vaccination, such as intramuscular, subcutaneous, or intradermal administration, and is compatible with existing vaccination protocols. It requires no special equipment or technical training, facilitating rapid deployment and application by medical institutions. Furthermore, its excellent immunogenicity and protective efficacy make it suitable for both pre-exposure prophylaxis in healthy individuals and post-exposure emergency prevention, thus enabling its wide range of applications.

[0040] In summary, the novel adjuvant subunit rabies virus vaccine of this invention exhibits significant advantages in terms of immunization efficacy, safety, ease of storage and transportation, and production cost, providing a more reliable and universally applicable technical option for rabies prevention and possessing important public health value. Attached Figure Description

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0042] Figure 1 The characteristics of the LNP-mRNA-G-H270P vaccine described in Comparative Example 1 are shown; wherein, (A) the particle size of the LNP-mRNA-G-H270P vaccine; (B) the polydispersity index (PDI) of the LNP-mRNA-G-H270P vaccine; (C) the encapsulation efficiency of the LNP-mRNA-G-H270P vaccine; and (D) the integrity of the LNP-mRNA-G-H270P vaccine was determined by 1% denaturing agarose gel electrophoresis.

[0043] Figure 2 The titer of RABV-G specific IgG in the serum of mice in Test Example 1 was detected by enzyme-linked immunosorbent assay (ELISA); (A) IgG titer 7 days after the first immunization; (B) IgG titer 14 days after the first immunization; (C) IgG titer 42 days after the first immunization; (D) Changes in IgG titer over time before and after the first immunization.

[0044] Figure 3 The results of the rabies virus neutralizing antibody titer in Test Example 2, as detected by the RFIIT method, are shown.

[0045] Figure 4The results of the enzyme-linked immunospot (ELISPOT) assay of spleen cells in Test Example 3 are shown; (A) spleen cells that produced IFN-γ after RABV-G stimulation; (B) spleen cells that produced IL-2 after RABV-G stimulation; (C) representative images of spleen cells that produced IFN-γ; (D) representative images of spleen cells that produced IL-2; the number of ELISPOT assays was determined by one-way ANOVA and Dunnett's multiple comparison test, with the G+QS21+CpG vaccine group as the control group; *p<0.05. **p<0.01. ****p<0.0001;

[0046] Figure 5 The results of flow cytometry analysis of CD4+ T cells expressing RABV-G-specific T helper 1 (Th1) cytokines and the supernatant of spleen cell culture for RABV-G-specific expression are shown in Test Example 4. (A) The proportion of CD4+ T cells producing IFN-γ in spleen cells after RABV-G stimulation; (B) The proportion of CD4+ T cells producing IL-2 in spleen cells after RABV-G stimulation; (C) A pseudo-color image providing a visual representation of the average expression levels of IFN-γ (Q1+Q2) and IL-2 (Q3+Q2) in a specific CD4+ T cell population. The concentrations of IFN-γ (D) and IL-2 (E) secreted by spleen cells after stimulation with 10 μg / mL RABV-G were measured using ELISA. Data were analyzed using one-way ANOVA and Dunnett's multiple comparison test, with the G+QS21+CpG vaccine group as the control group. *p<0.05. **p<0.01. ns, no significant difference;

[0047] Figure 6 The survival rate and weight changes of mice after viral challenge in Test Example 5 are shown; (A) the weight of surviving mice was recorded and the average daily weight (mean ± standard deviation, n = 10) was calculated over 14 days post-infection; (B) the Kaplan-Meier survival curves of the percentage of mice surviving over 14 days post-infection are shown; each group of mice was inoculated with CVS-11 at a dose of 50 LD50 / mouse (n = 10 per group). Detailed Implementation

[0048] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0049] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0050] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0051] In the following examples, CpG 1018S was purchased from Shanghai Sangon Biotech, QS21 was purchased from AlphaDiagnoestic International, and the aluminum hydroxide adjuvant was Alhydrogel, purchased from Croda (CAS: 21645-51-2).

[0052] Example 1

[0053] This embodiment provides a novel adjuvant subunit rabies virus vaccine, which comprises an antigen and an adjuvant; the antigen is the extracellular domain of a recombinantly expressed rabies virus (RABV) glycoprotein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0054] RABV-G-Etc:

[0055] MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCT

[0056] NLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFR

[0057] PTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVA

[0058] DLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFT

[0059] NSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAM

[0060] QTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSV

[0061] SFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGG

[0062] RCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPS

[0063] TVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCWRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSGGETGL.

[0064] The adjuvant is a combination of CpG oligodeoxynucleotide and QS21.

[0065] The method for preparing the novel adjuvant subunit rabies virus vaccine includes the following steps:

[0066] (1) The extracellular domain of RABV glycoprotein (RABV-G) was prepared by gene recombination and purified for later use;

[0067] (2) The purified RABV-G and adjuvants QS21 and CpG oligodeoxynucleotides were mixed evenly in sterile phosphate-buffered saline to obtain the subunit rabies virus vaccine (G+QS21+CpG).

[0068] Each dose of vaccine contains 5 μg of antigen RABV-G, 5 μg of adjuvant QS21, and 10 μg of adjuvant CpG oligodeoxynucleotides.

[0069] Comparative Example 1: mRNA Rabies Virus Vaccine

[0070] This comparative example provides an mRNA rabies virus vaccine, comprising an mRNA sequence encoding the extracellular domain of a RABV glycoprotein with an H270P targeting mutation. The mRNA is encapsulated with LNPs to prepare an LNP-mRNA-G-H270P formulation for use as a comparative example. The preparation method is as follows:

[0071] The synthesized DNA sequence was transcribed into an mRNA sequence encoding the extracellular domain of a RABV glycoprotein with an H270P targeting mutation via in vitro transcription. After purification with magnetic beads, the mRNA concentration was measured and dissolved in citrate-sodium citrate buffer (0.1M, pH 4). The mRNA vaccine was then encapsulated with self-made lipid nanoparticles (LNP) to prepare a 15.4 μg / animal / injection mRNA rabies virus vaccine (LNP-mRNA-G-H270P).

[0072]

[0073] Key physicochemical properties of LNP-mRNA-G-H270P were rigorously evaluated, including LNP particle size, polydispersity index (PDI), mRNA integrity, and encapsulation efficiency.

[0074] Test results as follows Figure 1 As shown, the average particle size of LNP-mRNA-G-H270P is 94.1 nm. Figure 1 (A) meets the requirements. The average PDI measurement value is 0.135 ( Figure 1 (B) indicates that the LNP particles are highly uniform in size. The encapsulation efficiency was measured to be 93.25%. Figure 1 (C) indicates effective packaging of mRNA. Integrity was assessed using formaldehyde-denaturing agarose gel electrophoresis. Figure 1 The D-band (indicated by a single band) shows good integrity. In summary, these results indicate that the mRNA was successfully encapsulated in LNPs.

[0075] Comparative Example 2: Aluminum Adjuvant Rabies Virus Vaccine

[0076] This comparative example provides an aluminum adjuvant rabies virus vaccine prepared via an antigen adsorption process. Specifically, the purified RABV glycoprotein extracellular domain (RABV-G) (5 μg per dose) prepared in Example 1 was continuously rotated with aluminum hydroxide adjuvant (1 mg aluminum per dose) at 4°C for 12 hours to ensure adequate antigen adsorption, yielding the aluminum adjuvant rabies virus vaccine (G+Alum).

[0077] Animal experiments:

[0078] To compare the immune levels and protective effects of four rabies virus vaccines, the following animal experiments were conducted.

[0079] Experimental animals: 4-week-old female BALB / c mice (weighing 14-16 grams) were housed in a specific pathogen-free (SPF) environment at the Small Animal Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences (IMBCAMS) and randomly divided into 5 groups of 15 mice each.

[0080] Positive control group: Received 1 / 6 of the dose of commercially available inactivated rabies virus vaccine (Ningbo Rong'an Biopharmaceutical Co., Ltd., Ningbo, China, labeled potency ≥2.5 IU / dose).

[0081] Negative control group: Administered an equal volume of PBS.

[0082] The other three experimental groups were injected intramuscularly with 50 μL of different antigens, including the G+QS21+CpG vaccine group described in Example 1, the LNP-mRNA-G-H270P vaccine group described in Comparative Example 1, and the G+Alum vaccine group described in Comparative Example 2.

[0083] A booster immunization was administered 4 weeks after the initial immunization, at the same dose. Blood samples were collected on days 7 and 14 after the initial immunization, and 2 weeks after the final immunization, to measure antibody titers and neutralizing antibody levels. Spleens were then harvested for cellular immunoassay.

[0084] The challenge experiment was conducted two weeks after final immunization. Specifically, two weeks after final immunization, each group of mice was injected intracranially with 30 μL of rabies virus strain CVS-11 (Challenge Virus Standard-11) with a viral titer of 50 LD50. The mice were observed for 14 consecutive days, and their survival rate and weight changes were recorded.

[0085] Test Example 1:

[0086] Serum samples collected from immunized mice were analyzed for the levels of antibodies specific to rabies virus glycoprotein (RABV-G) using indirect ELISA. One-way ANOVA and Dunnett's multiple comparison test were performed, with the G+QS21+CpG vaccine group serving as the control group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. No significant difference was observed within ns. The procedure was briefly as follows: Recombinant rabies virus glycoprotein was expressed in mammalian cells with a purity greater than 90% (confirmed by SDS-PAGE) in monomeric form (predicted molecular weight 60.01 kDa). It was diluted with PBS to a final concentration of 2 μg / mL and used to coat 96-well microplates. After overnight incubation at 4°C, unbound antigen was removed by washing three times with PBST (PBS containing 0.05% (v / v) Tween-20). Non-specific binding sites were blocked in PBS with 5% (w / v) skim milk powder at room temperature for 1 hour. Mouse serum was subjected to a series of two-fold dilutions (ranging from 1:2,000 to 1:4,096,000) and incubated at 37°C for 1 hour. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (dilution ratio 1:10,000) was used as the secondary antibody. After incubation, the reaction was terminated by adding 3,3′,5,5′-tetramethylbenzidine (TMB) for 5 minutes, followed by the addition of 2 mol / L sulfuric acid. The absorbance (OD450) was measured at 450 nm using a microplate reader. Antibody titers were determined based on an OD450 value ≥0.15 (cutoff threshold) at the highest serum dilution. Samples with OD450 values ​​below the cutoff value at the initial dilution (1:2,000) were assigned a titer of 100 in the statistical analysis.

[0087] Figure 2The results showed that on day 7 after the first immunization, the G+QS21+CpG vaccine group (5620), the LNP-mRNA-G-H270P vaccine group (3000), and the commercially available inactivated rabies virus vaccine group (1440) rapidly induced antigen-specific IgG antibody titers, while no IgG response was detected in the G+Alum vaccine group. Due to the low overall antibody levels at this early time point, there were no statistically significant differences between the groups. Figure 2 (A). By day 14, IgG titers had significantly increased in all vaccination groups. Figure 2 (B). Notably, the LNP-mRNA-G-H270P group achieved the highest rabies virus G protein-specific IgG titer (40,000), which was 1.25 times higher than the G+QS21+CpG group (32,000; p = 0.5953), 25 times higher than the G+Alum vaccine group (1,600; p < 0.001), and 16.7 times higher than the inactivated vaccine group (2,400; p < 0.01). Before the challenge, final antibody measurements showed that most vaccine groups maintained high levels of IgG ( Figure 2 (C). The G+QS21+CpG group achieved the highest rabies virus G protein-specific IgG titer (2,918,400), which was 3.08 times higher than the LNP-mRNA-G-H270P group (947,200; p<0.01), 45.6 times higher than the G+Alum group (64,000; p<0.001), and 145.77 times higher than the inactivated vaccine group (20,020; p<0.0001). Dynamic IgG analysis revealed different immune response patterns among the groups. Figure 2 (D). Both the G+QS21+CpG vaccine group and the LNP-mRNA-G-H270P vaccine group showed rapid seroconversion, high peak titers, and continuously increasing antibody levels over time. In contrast, the G+Alum vaccine group showed a delayed IgG response and lower overall titers, while the inactivated vaccine group tended to plateau at later time points after early induction.

[0088] Test Example 2:

[0089] The neutralizing antibody response of immunized mouse serum to rabies virus was assessed using the Rapid Fluorescent Focus Inhibition Assay (RFFIT) according to standard WHO protocols. A simplified procedure was as follows: serum samples were first inactivated by heating at 56°C for 30 minutes, followed by analysis. Human rabies immunoglobulin (HRIG, national reference standard) was used as the quantitative reference standard. For the experiment, the challenge virus standard (CVS-11 strain) was titrated to 10 μL / mL. 6 One fluorescent focal unit (FFU). BSR cells (passage 107-117) were used at 1 × 10⁶ cells per well. 6Cell density preparation. Serum samples were serially diluted threefold in 96-well plates (range 1:3 to 1:59,049). Each diluted serum sample was mixed with an equal volume of virus suspension and incubated at 37°C, 5% CO2 for 90 min to allow for virus neutralization. After neutralization, BSR cells were added to each well and cultured under the same conditions for 24 h. After incubation, cells were washed with PBS and fixed with 80% cold acetic acid. Immunofluorescence staining was performed using FITC-labeled anti-rabies monoclonal antibody (1:50 dilution in PBS) at 37°C for 30 min. After thorough washing of PBS, fluorescence focus was examined and quantified using a Leica DMI8 fluorescence microscope. To ensure reliability, all experiments were repeated and scored by two independent investigators. Neutralizing antibody titers were calculated using the Reed and Muench method, expressed as International Units per mL (IU / mL), rounded to two decimal places. This standardized method allowed for accurate comparison of neutralizing antibody levels in different serum samples.

[0090] RFFIT results revealed significant differences in RVNA titers among different vaccine groups. Figure 3 The G+QS21+CpG vaccine group produced the highest RVNA titer (2246 IU / mL), showing 3-fold (806 IU / mL, p<0.0001), 38-fold (59 IU / mL, p<0.0001), and 115-fold (20 IU / mL, p<0.0001) higher neutralizing activity compared to the LNP-mRNA-G-H270P vaccine group, the G+Alum vaccine group, and the commercially available inactivated rabies virus vaccine group, respectively. No response was detected in the PBS control group, confirming the validity of the experiment.

[0091] Test Example 3:

[0092] Spleens were mechanically separated using a 40-micron cell sieve and treated with ammonium chloride-potassium (ACK) lysis buffer for 5 minutes at room temperature to remove erythrocytes. The separated spleen cells were then suspended in complete RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum and penicillin-streptomycin at a density of 3 × 10⁶ cells / mL. 6 Cells were seeded. For ELISPOT analysis, 100 μL of cell suspension was seeded into each well of a 96-well plate. ELISPOT assays were performed using a commercial kit according to the manufacturer's instructions. To assess specific T-cell responses to rabies virus G protein, spleen cells were stimulated with 20 μg / mL of recombinant rabies virus G protein and incubated overnight. Following immunostaining, spot formation was quantified using an automated ELISPOT reading system.

[0093] ELISPOT analysis showed that the G+QS21+CpG vaccine group produced 78 spots under RABV-G stimulation, a significant increase of 15.6 times compared to the PBS group (5 spots, p<0.05). The LNP-mRNA-G-H270P vaccine group (each 3×10⁻⁶ spots) also showed a significant increase. 5 The spleen cells produced 166 spots, indicating more IFN-γ secreting spleen cells than the G+QS21+CpG vaccine group. Figure 4 (A). Regarding RABV-G specific IL-2 secreting spleen cells ( Figure 4 In the PBS group (8 spots, 10.88-fold lower, p<0.0001), the G+QS21+CpG vaccine group produced 87 spots, significantly higher than the PBS group (8 spots, 10.88-fold lower). In contrast, the LNP-mRNA-G-H270P vaccine group further increased the number of spots by 1.66-fold (144 spots) compared to the G+QS21+CpG vaccine group (p<0.01), both statistically significant. In repeated independent experiments, spleen cells from both the G+QS21+CpG and LNP-mRNA-G-H270P vaccine groups produced significantly more cytokine-secreting cells compared to the PBS group, as reflected in the dense, well-defined spot formation. Figure 4 (C and D in the middle).

[0094] Test Example 4:

[0095] Flow cytometry analysis was performed to prepare single-cell suspensions from harvested spleen cells at a concentration of 1 × 10⁻⁶ cells / mL. 7 Cells were seeded at a density of 2 × 10⁶ cells per well in 24-well plates. 6 Cells were stimulated with recombinant G protein (10 μg / mL) at 37°C and 5% CO2 for 2 hours. Bleomycin A was then added to inhibit extracellular cytokine secretion, followed by an additional overnight incubation. After 16 hours, cells were collected and viability stained. Cells were then fixed, permeabilized, and stained intracellularly with PE-labeled anti-mouse IFN-γ antibody and APC-labeled anti-mouse IL-2 antibody. Cell percentage was analyzed using flow cytometry.

[0096] according to Figure 5 The flow cytometry analysis results shown are similar to those obtained by ELISPOT. Following RABV-G stimulation, flow cytometry analysis revealed that the proportion of CD4+ T cells producing IFN-γ in the G+QS21+CpG vaccine group was 0.039%, which was 1.95 times higher than that in the PBS group (0.020%, p = 0.7949). Figure 5(A). The LNP-mRNA-G-H270P vaccine group showed stronger cellular immunity, with the proportion of CD4+ T cells produced by IFN-γ reaching 0.140%, which was 3.59 times higher than the baseline of the G+QS21+CpG vaccine group (p<0.05). Similarly, in terms of IL-2 expression ( Figure 5 In the PBS group (B), the G+QS21+CpG vaccine group induced a CD4+ T cell response of 0.040% (2.11-fold higher than the 0.019% in the PBS group, p = 0.7459). The LNP-mRNA-G-H270P vaccine group again showed stronger performance (0.149%), with an IL-2 response 3.7-fold higher than the G+QS21+CpG vaccine group (p < 0.01). All comparisons showed statistically significant differences. Figure 5 The study summarized the cytokine profile, confirming that the vaccine formulations (G+QS21+CpG vaccine group and LNP-mRNA-G-H270P vaccine group) not only enhanced IFN-γ production but also promoted IL-2 secretion, indicating a multifunctional T cell response. The positive control group showed a strong signal response, validating the reliability of the experimental system. Meanwhile, the PBS group showed a weak background response, effectively excluding non-specific interference.

[0097] Further cytokine analysis was performed by suspending spleen cells in complete RPMI 1640 medium containing 10% (v / v) fetal bovine serum and penicillin-streptomycin at a cell density of 1×10⁶ cells / mL. 7Cells / mL. 100 μL of cell suspension was dispensed into 96-well plates. For the positive control, cells were stimulated with 10 μL of PMA + iomycin (stock solution concentration: 500 ng / mL + 10 μg / mL). Experimental wells were stimulated with 10 μg / mL of G protein. After incubation at 37°C and 5% CO2 for 24 hours, the culture supernatant was collected for cytokine quantification. To detect cytokines, 96-well plates were coated overnight at 4°C with anti-IL-2 (3 μg / mL) or anti-IFN-γ (4 μg / mL) capture antibodies, and then blocked at 37°C for 1 hour with 5% (w / v) skim milk. 50 μL / well of the supernatant sample was incubated at room temperature for 3 hours with a standard curve generated using recombinant mouse IL-2 and IFN-γ. Subsequent assays used biotin-labeled anti-IL-2 or anti-IFN-γ antibodies (2 μg / mL) and HRP-labeled streptavidin (1 μg / mL), with each step involving incubation for 1.5 hours. Cytokine concentrations were measured by ELISA. Cytokine analysis following RABV-G stimulation revealed significant immunological differences between groups. The G+QS21+CpG vaccine group exhibited cytokine production, with IFN-γ levels of 63.13 ng / mL, significantly higher than the PBS control group (8.71 ng / mL), a 7.25-fold increase (p<0.01). The LNP-mRNA-G-H270P vaccine group showed moderately enhanced IFN-γ secretion (67.89 ng / mL, 1.08-fold higher than the G+QS21+CpG vaccine group, p = 0.8929). More notably, IL-2 secretion showed a significant increase. The G+QS21+CpG vaccine group (2.78 ng / mL) was 5.67 times higher than the PBS group (0.49 ng / mL, p = 0.7345), while the LNP-mRNA-G-H270P vaccine group reached 18.71 ng / mL, which was 6.73 times higher than the baseline of the G+QS21+CpG vaccine group (p < 0.01). Figure 5 middle DE).

[0098] Test Example 5:

[0099] Following vaccination and subsequent lethal challenge with 50 LD50 of the CVS-11 strain, all vaccine groups showed significantly improved outcomes compared to the PBS group. Figure 6 Weight dynamics ( Figure 6 As shown in Figure A), during the 14-day observation period, the body weight of mice in the G+QS21+CpG vaccine group and the LNP-mRNA-G-H270P vaccine group remained stable, indicating minimal clinical impact from the infection. In contrast, the G+Alum vaccine group and the commercially available inactivated rabies virus vaccine group experienced a transient decrease in body weight (8-12%) from day 5 to day 7 post-challenge, but all surviving animals returned to baseline body weight by day 10.

[0100] Survival analysis ( Figure 6 (B) The results showed that both the G+QS21+CpG vaccine group and the LNP-mRNA-G-H270P vaccine group achieved 100% protection, with the G+QS21+CpG vaccine group showing faster recovery of physiological parameters. The survival rate of the G+Alum vaccine group after challenge was 70%, comparable to the protective efficacy of the positive control group of the commercially available inactivated rabies virus vaccine. All PBS control mice died from infection on day 11, confirming the effectiveness of the challenge. Notably, the weight-survival correlation indicated that both the G+QS21+CpG vaccine group and the LNP-mRNA-G-H270P vaccine group not only prevented death but also maintained optimal health, as evidenced by the absence of significant weight fluctuations.

[0101] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A novel adjuvanted subunit rabies vaccine, characterized in that, The vaccine comprises an antigen and an adjuvant; The antigen is a rabies virus (RABV) glycoprotein extracellular domain; The adjuvant is a combination of CpG oligodeoxynucleotide and QS21.

2. The rabies virus vaccine according to claim 1, characterized in that, The RABV glycoprotein extracellular domain is a recombinantly expressed RABV-G protein extracellular segment, and its amino acid sequence is shown as SEQ ID NO.

1.

3. The rabies virus vaccine according to claim 1, characterized in that, The mass ratio of CpG oligodeoxynucleotide and QS21 in the adjuvant is 1-3:

1.

4. The rabies virus vaccine according to claim 1, characterized in that, The concentration of the antigen in the vaccine is 5 μg / needle, and the total amount of the adjuvant added is 15 μg / needle, wherein the mass ratio of CpG oligodeoxynucleotide and QS21 is 2:

1.

5. A process for the preparation of the novel adjuvanted subunit rabies vaccine according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: (1) preparing the RABV glycoprotein extracellular domain by using a genetic recombination method, and reserving after purification; (2) mixing the purified RABV glycoprotein extracellular domain with the adjuvant QS21 and CpG oligodeoxynucleotide uniformly in sterile phosphate buffered physiological saline to obtain the subunit rabies virus vaccine.

6. Use of a new adjuvanted subunit rabies vaccine according to any one of claims 1 to 4, characterized in that, The application comprises: 1) preparing a pharmaceutical composition for preventing or treating diseases caused by infection of the rabies virus; 2) preparing a pharmaceutical composition for preventing or treating infection of the rabies virus.

7. Use according to claim 6, characterized in that, The pharmaceutical composition is administered by a muscle, subcutaneous or intradermal injection route.

8. Use according to claim 6, characterized in that, The pharmaceutical composition is used for inducing the body to produce specific IgG antibodies and virus neutralizing antibodies against RABV, and inducing a Th1 type cellular immune response.

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