A rabies mRNA-lnp vaccine enhanced by nanose and a preparation method thereof

Abstract

The application discloses a kind of rabies mRNA-LNP vaccine enhanced by nano selenium and a preparation method thereof, belong to biological medicine, vaccine preparation and nanometer material application technical field.The vaccine takes mRNA coding rabies virus glycoprotein as antigen component, takes SeNPs as immune enhancement component, and realizes the synergistic delivery of both through LNP delivery system.Adopt fishbone-shaped microfluidic chip to prepare the SeNPs of uniform particle size, and further construct the core-shell type nano vaccine structure with SeNPs as core and LNP as outer layer.The obtained vaccine has the characteristics of uniform particle size, good dispersity and high encapsulation efficiency, and can effectively promote mRNA delivery and antigen expression.Compared with prior art, the application utilizes the synergistic effect of SeNPs and mRNA vaccine, significantly improves the immunogenicity of rabies mRNA vaccine, including significant enhancement of antigen-specific antibody response and virus VNA level.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, vaccine preparation and nanomaterial application technology, and in particular to a rabies mRNA-LNP vaccine enhanced with nano-selenium and its preparation method. Background Technology

[0002] Rabies is a highly fatal zoonotic infectious disease caused by the rabies virus (RABV). Once symptoms appear, the mortality rate is close to 100%, and there is currently no effective treatment, posing a serious threat to public health. Vaccination remains the only feasible method for rabies prevention. The rabies virus genome encodes five structural proteins: nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), glycoprotein (G protein), and RNA-dependent RNA polymerase (L protein). Among these, the G protein is the only exposed protein on the viral surface and is the main antigen that induces the body to produce virus-neutralizing antibodies (VNA) and exerts a protective immune effect. Rabies immunoprotection is highly dependent on VNA; the World Health Organization (WHO) considers a rabies virus VNA titer ≥0.5 IU / mL as the effective protective threshold. Traditional rabies vaccines mainly include inactivated vaccines and live attenuated vaccines. Although they offer some protection, they still suffer from problems such as long production cycles, limited immunogenicity, short duration of immunity, and the need for multiple booster immunizations. In recent years, mRNA vaccines have attracted widespread attention due to their advantages such as flexible design, short production cycle, high safety, and ability to induce humoral and cellular immunity. The application of lipid nanoparticles (LNP) delivery systems has further improved the stability and in vivo delivery efficiency of mRNA. However, current rabies mRNA vaccines still suffer from insufficient immunogenicity, limited antibody duration, and low activation efficiency of the body's innate immunity, limiting their further application. Selenium nanoparticles (SeNPs), as a novel nanomaterial, possess good biocompatibility, low toxicity, and immunomodulatory activity, and can promote antigen presentation and enhance the body's immune response. Currently, SeNPs have been used in various vaccine adjuvant studies, but research on the co-delivery of SeNPs with rabies mRNA vaccines is limited, especially lacking an LNP vaccine system capable of simultaneously encapsulating SeNPs and rabies virus mRNA. Therefore, developing an LNP vaccine capable of co-delivering SeNPs and rabies virus mRNA to improve the stability and immunogenicity of mRNA vaccines is of great significance for enhancing the immunogenicity and application value of rabies vaccines. Summary of the Invention

[0003] The purpose of this invention is to address the problems of insufficient immunogenicity and the need for multiple injections in existing rabies mRNA vaccines by providing a nano-selenium-enhanced rabies mRNA-LNP vaccine and its preparation method, so as to improve the humoral immunity level induced by rabies mRNA vaccine and achieve single-injection immunization.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a rabies mRNA-LNP vaccine enhanced with nano-selenium, comprising the following steps: Na2SeO3 solution and ascorbic acid solution were mixed and then aged to obtain selenium nanoparticles. A recombinant vector containing the rabies virus glycoprotein G encoding gene was constructed, and the recombinant vector was linearized to obtain a linearized plasmid template; the accession number of the rabies virus glycoprotein G encoding gene is M32751.1; Using the linearized plasmid as a template, mRNA encoding rabies virus glycoprotein G was synthesized through in vitro transcription, capping, and tailing reactions. The citrate buffer containing the SeNPs particles and the rabies virus mRNA was rapidly mixed with the ethanol phase containing lipid components using a microfluidic mixing method to form lipid nanoparticles, which were then purified to obtain a selenium-enhanced rabies mRNA-LNP vaccine.

[0005] Optionally, the volume ratio of the Na2SeO3 solution to the ascorbic acid solution is 1:1; the concentration of Na2SeO3 in the Na2SeO3 solution is 10 mM; and the concentration of ascorbic acid in the ascorbic acid solution is 40 mM.

[0006] Optionally, the ripening time is 0.5-24 h.

[0007] Optionally, the final concentration of the SeNPs particles in the citrate buffer containing the SeNPs particles and the rabies virus mRNA is 10 μg / mL, and the final concentration of the rabies virus mRNA is 100 μg / mL.

[0008] Optionally, the lipid component includes ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphatidylcholine, cholesterol, and PEG-lipid.

[0009] Optionally, the molar ratio of the ionizable lipid, the 1,2-distearate-sn-glycerol-3-phosphatidylcholine, the cholesterol, and the PEG-lipid is 50:10:38.5:1.5.

[0010] More preferably, the ionizable lipid is SM102.

[0011] Optionally, the volume ratio of the citrate buffer containing the SeNPs particles and the rabies virus mRNA to the ethanol phase containing lipid components is 3:1.

[0012] This invention provides a rabies mRNA-LNP vaccine prepared using the above method.

[0013] This invention provides the application of nano-selenium as an adjuvant in rabies mRNA vaccines.

[0014] Optionally, the method for preparing the nano-selenium includes the step of mixing Na2SeO3 solution and ascorbic acid solution and then aging them to obtain the nano-selenium.

[0015] The present invention discloses the following technical effects: This invention improves a nano-selenium-enhanced rabies mRNA-LNP vaccine. This vaccine uses mRNA encoding rabies virus glycoproteins as the antigenic component and SeNPs as the immune-enhancing component, achieving synergistic delivery of both via an LNP delivery system. In the preparation of the nano-selenium-enhanced rabies mRNA-LNP vaccine, this invention utilizes a fishbone-shaped microfluidic chip to rapidly prepare uniformly sized SeNPs; further, it constructs a core-shell LNP structure for co-delivery of the adjuvant SeNPs and the mRNA vaccine. In other words, this invention constructs a core-shell nanovaccine structure with SeNPs as the core and LNPs as the outer layer. This invention uses SeNPs as an adjuvant for the rabies mRNA vaccine, significantly enhancing the immunogenicity of the rabies mRNA vaccine. This vaccine possesses a uniform, regular, and stable nanostructure, high encapsulation efficiency, and good dispersion stability, effectively promoting mRNA delivery and antigen expression, laying the foundation for subsequent in vivo evaluation of immunization efficacy. Compared with existing technologies, this invention utilizes the synergistic effect of SeNPs and mRNA vaccines to significantly improve the immunogenicity of rabies mRNA vaccines, including a significant enhancement of antigen-specific antibody responses and VNA levels. The preparation process of this invention is simple and reproducible, demonstrating promising application prospects and industrialization value.

[0016] Furthermore, this invention also reveals that SeNPs are not only highly compatible with the mRNA-LNP system, but also significantly enhance the in vitro antigen expression capacity of rabies mRNA vaccines. Simultaneously, SeNPs significantly enhance the humoral immune response induced by rabies mRNA vaccines and exhibit a trend of promoting sustained enhancement of antibody responses, suggesting their potential application value as adjuvants for mRNA vaccines. This invention provides an important foundation for further enhancing vaccine immunogenicity. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A schematic diagram of SeNPs fabricated using a microfluidic chip; Figure 2 The image shows the appearance of the prepared SeNPs solution; Figure 3 Image of SeNPs particles obtained by transmission electron microscopy; Figure 4 Figure showing the particle size distribution and dispersion coefficient of SeNPs; Figure 5 The result is a graph showing the Zeta potential of SeNPs particles; Figure 6 Image of high-resolution transmission electron microscopy results for SeNPs particles; Figure 7 The image shows the results of energy dispersive X-ray spectroscopy (EDX) analysis of Se in SeNPs particles. Figure 8 A schematic diagram of the SeNPs-LNPs particle structure; Figure 9 The image shows the appearance of the prepared LNP-SeNPs & mRNA-G vaccine solution. Figure 10 Transmission electron microscopy (TEM) results of LNP-SeNPs & mRNA-G vaccine nanoparticles; Figure 11 The image shows the encapsulation rate of LNP-SeNPs & mRNA-G vaccines; where LNP-mRNA-G: LNP-mRNA-G vaccine without SeNPs; LNP-SeNPs & mRNA-G: LNP-mRNA-G vaccine with SeNPs. Figure 12 Figure showing the particle size distribution of LNP-SeNPs & mRNA-G nanoparticles; Figure 13 The figure shows the zeta potential results of LNP-SeNPs & mRNA-G nanoparticles; where green represents the zeta potential of LNP-SeNPs & mRNA-G particles at pH 7.4; and purple represents the zeta potential of LNP-SeNPs & mRNA-G particles at pH 4.0. Figure 14Immunofluorescence results of G protein expression levels in HEK-293T cells for LNP-mRNA-G vaccine (A) and LNP-SeNPs&mRNA-G vaccine (B); Figure 15 The graph shows the results of rabies virus IgG antibody titer detection; where A represents week 2 and B represents week 5. Figure 16 The graph shows the results of rabies virus VNA titer testing; where A represents week 2 and B represents week 5. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Construction and characterization of SeNPs 1.1 Preparation of 10 mM sodium selenite (Na2SeO3) solution Weigh 0.173 g of anhydrous Na₂SeO₃ (molecular weight 172.94 g / mol) into an RNase-free centrifuge tube, add 80 mL of RNase-free ultrapure water to dissolve it completely, then bring the volume to 100 mL with RNase-free ultrapure water and mix well to obtain a 10 mM Na₂SeO₃ solution. The obtained solution can be stored at 4℃ protected from light for later use.

[0025] 1.2 Preparation of 40 mM ascorbic acid solution Weigh 0.704 g of ascorbic acid (vitamin C, Vc, molecular weight 176.12 g / mol) into an RNase-free centrifuge tube, add 80 mL of RNase-free ultrapure water to dissolve it completely, vortex until completely dissolved, and then bring the volume to 100 mL with RNase-free ultrapure water. Mix thoroughly to obtain a 40 mM Vc solution. Since Vc is easily oxidized, this solution should be prepared and used immediately.

[0026] 1.3 Preparation of SeNPs Particles SeNPs were prepared using a microfluidic chip-assisted chemical reduction method. A 10 mM Na₂SeO₃ solution and a 40 mM Vc solution were injected into two independent inlet channels of a fishbone-shaped microfluidic chip (manufacturer: Shanghai Pengzan Biotechnology Co., Ltd.; catalog number: PDMS-LNP-B0) at room temperature with an equal volume ratio (1:1, v / v). The two phases were rapidly and uniformly mixed within the chip through laminar diffusion and mixing structural units, resulting in a reduction reaction. A consistent flow rate was maintained to ensure stable hydrodynamic conditions. After mixing, the resulting reaction system was aged at room temperature. The aging process included standing, gentle agitation, or low-speed stirring, with standing aging being preferred. The aging time was 0.5–24 h, preferably 2–4 h; in this example, the aging time was 2.5 h. The aging process allowed the selenium precursor to react fully, promoting the formation of SeNPs particles and improving their particle size distribution uniformity and colloidal stability. After maturation, the reaction system was centrifuged at 12,000 rpm for 15 min, and the SeNPs precipitate was collected. The precipitate was washed 2-3 times with RNase-free ultrapure water to remove unreacted precursors, reducing agents, and other impurities. The washed SeNPs precipitate was resuspended in RNase-free ultrapure water to obtain a SeNPs particle dispersion. The obtained SeNPs particle dispersion was stored at 4°C in the dark for later use.

[0027] 1.4 Characterization of SeNPs particles The prepared SeNPs particles were characterized for their morphology, particle size distribution, surface charge, and elemental composition. Transmission electron microscopy (TEM) was used to observe the microstructure of the SeNPs particles. A nanoparticle size and zeta potential analyzer was used to determine the hydrodynamic particle size, polydispersity index (PDI), and zeta potential of the nanoparticles to assess their particle size distribution uniformity, surface charge characteristics, and colloidal stability. Simultaneously, high-resolution transmission electron microscopy (HRTEM) was used to observe the fine structure of the nanoparticles, and energy-dispersive X-ray spectroscopy (EDX) was combined with this method to analyze their elemental composition, confirming the presence of selenium in the nanoparticles. All the above tests were performed according to the instrument's standard operating procedures.

[0028] 2. Preparation of rabies mRNA-G vaccine The pcDNA3.1-G plasmid was constructed using the G protein gene sequence of the rabies virus HEP-Flury strain (GenBank: AB085828.1, accession number M32751.1). Specifically, the pcDNA3.1 plasmid was used as the base plasmid, with Nhe I and BamHI as restriction enzyme sites, and the HEP-Flury strain G protein gene sequence (accession number M32751.1) was inserted to obtain the pcDNA3.1-G plasmid. Then, it was linearized by single digestion with BamHI enzyme and used as an in vitro transcription template. N1-methylpseudouridine was incorporated as a uridine substitute using the T7 high-yield RNA transcription kit. The linear DNA template used as the in vitro transcription template (the linearized pcDNA3.1-G plasmid) contained the antigen coding region, 5′ and 3′ untranslated regions (UTRs), a 5′ cap structure, and a poly(A) tail. Cap structures and poly(A) tails were added to the 5′ and 3′ ends of mRNA using the Cap1 capping kit and tailing kit from Nearshore Proteins, respectively. Purification of the mRNA product involved mixing with lithium chloride and incubating at -20°C for at least 30 min. Subsequently, the precipitate was collected by centrifugation at 12,000 × g for 15 min at 4°C. The precipitate was then washed three times with pre-chilled 70% ethanol and resuspended in RNase-free ultrapure water to obtain the rabies mRNA-G vaccine. The prepared mRNA was stored at -80°C for further use.

[0029] 3. Preparation of LNP-SeNPs & mRNA-G vaccines Microfluidic methods were used to prepare encapsulated mRNA and SeNPs. First, 50 mL each of 100 mM citric acid (molecular weight: 210.14, 1.05 g citric acid dissolved in ultrapure water) and 100 mM sodium citrate (molecular weight: 294.10, 1.47 g sodium citrate dissolved in ultrapure water) solutions were prepared using ultrapure water. 33.0 mL of the citric acid solution and 17.0 mL of the sodium citrate solution were mixed thoroughly, and DEPC was added to a final concentration of 0.1% (v / v). The mixture was then vigorously shaken for 10 min, allowed to stand for 30 min, and then autoclaved to remove DEPC. After sterilization, the solution was brought to a final volume of 100 mL with RNase-free ultrapure water to obtain a 50 mM citrate buffer (pH=4). The mRNA was added to the citrate buffer to a concentration of 100 μg / mL; simultaneously, the prepared SeNPs particles were added to the citrate buffer to a concentration of 10 μg / mL, forming the aqueous phase. The ionizable lipids (SM102), 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), cholesterol, and PEG-lipid were then mixed in a molar ratio of 50:10:38.5:1.5. The lipid mixture was dissolved in anhydrous ethanol to achieve a total lipid concentration of 12 mM, which constituted the ethanol phase. The ethanol and aqueous phases were then rapidly mixed in a microfluidic device at a volume ratio of 1:3. The prepared LNP-SeNPs&mRNA-G solution was diluted 30 times with 1×PBS buffer (pH 7.4) and then concentrated to an RNA concentration of 100 μg / mL using a 30 kD ultrafiltration tube to obtain the LNP-SeNPs&mRNA-G solution, i.e., the rabies LNP-SeNPs&mRNA-G vaccine. Finally, the LNP-SeNPs&mRNA-G solution was sterilized through a 0.22 µm filter and briefly stored at 4°C for later use.

[0030] 4. In vitro expression analysis of rabies LNP-SeNPs & mRNA-G vaccine The in vitro expression level of LNP-SeNPs & mRNA-G vaccine was evaluated using HEK-293T cells. HEK-293T cells were routinely cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. When the cell confluence reached 70%-80%, the cells were cultured at 1 × 10⁻⁶ cells / cells. 5 / wells were seeded in 24-well plates. After culturing for 24 h until cells adhered, LNP-SeNPs & mRNA-G vaccine containing 1 μg of rabies virus G protein-encoding mRNA (obtained by dilution of the LNP-SeNPs & mRNA-G vaccine prepared in step "3. Preparation of LNP-SeNPs & mRNA-G Vaccine") and LNP-mRNA-G vaccine without SeNPs (prepared using the same method as LNP-SeNPs & mRNA-G vaccine, except without SeNPs) were added. Untreated cells were set up as a negative control group. Cells were then incubated in serum-free DMEM medium. After 6 h of incubation, the medium was replaced with DMEM medium containing 2% fetal bovine serum (FBS). After another 24 h of incubation, the medium was discarded, and the cells were gently washed twice with PBS buffer, followed by fixation with 4% paraformaldehyde at room temperature for 15 min. After fixation, cells were gently washed three times with PBS buffer and permeabilized with 0.1% Triton X-100 for 10 min. After permeabilization, cells were blocked with PBS solution containing 5% BSA at room temperature for 30 min to reduce non-specific binding. Anti-DDDDK-tagged antibody (manufacturer: abcam; catalog number: ab18230) was then added as the primary antibody and incubated overnight at 4°C. The next day, cells were washed three times with PBS buffer and then incubated with FITC-labeled goat anti-mouse IgG secondary antibody (manufacturer: abcam; catalog number: ab6785) at room temperature in the dark for 1 h. After incubation, cells were washed again with PBS, and images were acquired using a fluorescence microscope. The in vitro antigen expression levels of different vaccine systems were evaluated by comparing the intensity of the green fluorescence signal and the number of positive cells.

[0031] 5. Humoral immune evaluation of rabies LNP-SeNPs & mRNA-G vaccine Female ICR mice aged 6-8 weeks were selected for animal immunization experiments. Mice were randomly divided into three groups: PBS group, LNP-mRNA-G vaccine group, and LNP-SeNPs&mRNA-G vaccine group, with 10 mice in each group. Each experimental group was immunized with a single injection. In the LNP-mRNA-G vaccine group and the LNP-SeNPs&mRNA-G vaccine group, each mouse was injected with a vaccine preparation containing 2 μg of rabies virus G protein-encoding mRNA (obtained by diluting the rabies mRNA-G vaccine prepared in step "2, Preparation of Rabies mRNA-G Vaccine" and the LNP-SeNPs&mRNA-G vaccine prepared in step "3, Preparation of LNP-SeNPs&mRNA-G Vaccine"). The PBS group received an equal volume of PBS as a negative control. Immunization was performed via intramuscular injection into the quadriceps femoris muscle region of the mouse's hind limb. The injection volume was 50-100 μL per dose; in this example, the injection volume was 50 μL. Serum samples were collected from mice at weeks 2 and 5 post-immunization for subsequent detection of rabies virus-specific IgG antibodies and VNA levels.

[0032] 6. Detection method for rabies virus-specific IgG antibodies The level of rabies virus-specific IgG antibodies in mouse serum was detected using an indirect ELISA method. Mouse serum was first collected and inactivated at 56°C for 30 min. The inactivated and purified rabies virus particles were diluted to 500 ng per well using coating buffer (5 mM Na2CO3, pH 9.6) and added to 96-well ELISA plates. The plates were incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were washed three times with PBST washing buffer (PBS containing 0.5% Tween-80). Then, 5% skim milk-PBS blocking buffer was added, and the plates were blocked for 6 h at 4°C. After blocking, the serum samples were diluted with PBST containing 5% skim milk, with the IgG detection serum diluted 1:2000. 100 μL of the diluted serum was added to each well, and the plates were incubated at 37°C for 2 h. After incubation, the liquid was discarded, and the plates were washed three times with PBST. Then, 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody was added to each well, and the plate was incubated at 37°C for 1 h. After the secondary antibody incubation, the plate was washed three times with PBST. Then, 100 μL of TMB chromogenic buffer was added to each well, and the plate was incubated at room temperature in the dark for approximately 15 min. Finally, 50 μL of 2 M H₂SO₄ was added to terminate the reaction. The absorbance (OD) of each well was finally measured at 450 nm using a microplate reader. 450 The IgG antibody level was used to evaluate the level of rabies virus-specific IgG antibodies in serum.

[0033] 7. Rabies virus VNA level detection The titer of rabies virus VNA was detected using a fluorescent antibody virus neutralization assay. Serum was obtained by centrifugation of mouse blood and inactivated at 56°C for 30 min. Subsequently, 100 μL of DMEM medium was added to each well of a 96-well cell culture plate. 50 μL of the test serum or rabies virus standard serum (0.5 IU / mL, purchased from the National Institute for Biological Standards and Control, Hertfordshire, UK) was added to the first column of wells, followed by serial 3-fold dilutions. Four replicates were set up for each sample. Then, 50 μL of 100 FFU of rabies virus CVS-11 suspension was added to each well, and the plate was incubated at 37°C for 1 h. After incubation, 2 × 10⁻⁶ cells / well were added to each well. 4 BSR cells were cultured at 37°C for 72 h. After culture, they were fixed in pre-cooled 80% acetone at room temperature for 30 min. After fixation, they were stained with FITC-labeled anti-rabies virus N protein antibody (manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd.; catalog number: 40913-MM02). After staining, the fluorescence signal was observed using an Olympus IX51 fluorescence microscope. The VNA titer was calculated by comparing the test serum with the reference standard serum. Rabies virus VNA titer is expressed in International Units per milliliter (IU / mL).

[0034] 8. Results and Analysis 8.1 Synthesis and Characterization of SeNPs Particles SeNPs particles were successfully prepared by rapidly mixing Na2SeO3 solution and Vc solution using a microfluidic chip with a fishbone-shaped structure via a redox reaction. Figure 1 The resulting SeNPs solution exhibits a uniform pink or brick-red color. Figure 2 The color of the particles was consistent with the characteristic color of classic SeNPs reported in the literature, initially indicating that the SeNPs particles had been successfully formed. Subsequently, the morphology and physicochemical properties of the SeNPs were systematically characterized. Transmission electron microscopy (TEM) results showed that the prepared SeNPs had a regular spherical structure, were uniformly dispersed, and no obvious agglomeration was observed. Figure 3 Dynamic light scattering (DLS) analysis showed that the average particle size of SeNPs was approximately 34.7 nm, and the polydispersity index (PDI) was 0.124, indicating that the system possesses good particle size uniformity and dispersion stability. Figure 4 Furthermore, the zeta potential of SeNPs is -28.32 mV, suggesting that they possess a certain degree of colloidal stability in aqueous systems. Figure 5To further confirm the microstructure and elemental composition of SeNPs, high-resolution transmission electron microscopy (HRTEM) combined with energy-dispersive X-ray spectroscopy (EDX) was used for analysis. HRTEM results showed that the obtained nanoparticles possess a dense nanostructure. Figure 6 EDX elemental analysis further confirmed that the SeNPs particles mainly contained Se element signals ( Figure 7 This indicates that the experiment successfully prepared SeNPs particles composed of selenium.

[0035] 8.2 Preparation and Characterization of Rabies LNP-SeNPs & mRNA-G Vaccine like Figure 8 As shown, the constructed LNP-SeNPs&mRNA-G vaccine delivery system mainly consists of a SeNPs&mRNA complex core and an outer LNP lipid membrane structure. First, the prepared SeNPs were thoroughly mixed with mRNA encoding the rabies G protein to form a complex system. Then, microfluidic technology was used to rapidly mix and encapsulate this complex with four LNP lipid components, ultimately successfully preparing an LNP-SeNPs&mRNA-G vaccine that simultaneously delivers SeNPs and mRNA. The resulting formulation exhibits a uniform pale yellow emulsion state. Figure 9 This indicates that the nanoparticle system has formed a stable structure. Subsequently, the morphology and physicochemical properties of the nanovaccine were systematically characterized. Transmission electron microscopy (TEM) results showed that the LNP-SeNPs&mRNA-G vaccine exhibited a regular spherical nanostructure with uniform particle dispersion and no obvious aggregation. Further analysis revealed a dense core structure with high electron density within the particles, surrounded by a complete membrane-like lipid layer. Figure 10 This indicates that SeNPs and mRNA have been successfully encapsulated by LNPs to form a stable nanodelivery system. Further analysis of its encapsulation performance revealed that the LNP-SeNPs&mRNA-G vaccine exhibits high encapsulation capacity for SeNPs and mRNA, with an overall encapsulation rate of 93.1%. Figure 11 This indicates that the delivery system can effectively load mRNA and SeNPs. Dynamic light scattering (DLS) analysis showed that the average particle size of LNP-SeNPs & mRNA-G was approximately 121.5 nm, and the polydispersity index (PDI) was 0.136. Figure 12 This indicates that the nanoparticle system possesses good particle size uniformity and dispersion stability, making it suitable for in vivo delivery applications. Furthermore, Zeta potential measurements showed that as the pH decreased from 7.4 to 4.0, the surface charge of LNP-SeNPs & mRNA-G underwent ionization changes, and the Zeta potential increased from -9.44 mV to +8.16 mV. Figure 13The presence of a near-neutral charge indicates that the system as a whole is in a state of reduced electrostatic aggregation between particles and improved colloidal stability in physiological environments. In summary, this invention successfully constructed an LNP nanodelivery system simultaneously loading SeNPs and rabies virus mRNA. This system possesses a regular and stable nanostructure, high encapsulation efficiency, and good dispersion stability, laying the foundation for subsequent in vivo evaluation of its immunogenicity.

[0036] 8.3 In vitro expression analysis of rabies LNP-SeNPs & mRNA-G vaccine To evaluate the in vitro expression capacity of the LNP-SeNPs & mRNA-G vaccine, HEK-293T cells were transfected. LNP-SeNPs & mRNA-G vaccine containing 1 μg of rabies virus G protein-encoding mRNA and LNP-mRNA-G vaccine without SeNPs were transfected into HEK-293T cells, respectively. Twenty-four hours after transfection, immunofluorescence staining with an anti-rabies virus G protein antibody was performed to detect the expression level of the G protein. Immunofluorescence results showed that both vaccines successfully expressed the rabies virus G protein in HEK-293T cells, but compared with the LNP-mRNA-G vaccine group, the LNP-SeNPs & mRNA-G vaccine group produced a stronger green fluorescence signal. Figure 14 The results indicate that the induced G protein expression level was significantly increased. Simultaneously, the number of positive expression cells in the LNP-SeNPs&mRNA-G group was significantly increased, indicating that the addition of SeNPs can effectively enhance the cellular delivery efficiency of mRNA. These results demonstrate that SeNPs are not only well compatible with the mRNA-LNP system but also significantly improve the in vitro antigen expression capacity of rabies mRNA vaccines, providing an important foundation for subsequently enhancing vaccine immunogenicity.

[0037] 8.4 Immunogenicity Analysis of Rabies LNP-SeNPs & mRNA-G Vaccine To evaluate the enhancing effect of SeNPs on humoral immune responses to rabies mRNA vaccines, mice were immunized with a single dose of 2 μg of LNP-SeNPs&mRNA-G vaccine and LNP-mRNA-G vaccine, with a PBS group serving as a negative control. Serum samples were collected from mice at weeks 2 and 5 post-immunization for the detection of IgG antibody and VNA levels. Results showed that no specific antibody response was detected in the PBS control group, while both the LNP-SeNPs&mRNA-G vaccine group and the LNP-mRNA-G vaccine group successfully induced significant IgG and VNA responses. Compared with the LNP-mRNA-G vaccine, the LNP-SeNPs&mRNA-G nanovaccine showed a significantly enhanced humoral immune response at all time points. Regarding IgG antibody levels, at week 2, the IgG titer in the LNP-SeNPs&mRNA-G group was approximately 3.8 times that of the LNP-mRNA-G group; by week 5, this difference had further increased to 11.6 times. Figure 15 Regarding VNA levels, the LNP-SeNPs&mRNA-G group was approximately 1.5 times that of the LNP-mRNA-G group at week 2, increasing to 2.3 times at week 5, and the VNA titer induced by the LNP-SeNPs&mRNA-G vaccine reached as high as 25.35 IU / mL at week 5. Figure 16 In conclusion, SeNPs can significantly enhance the humoral immune response induced by rabies mRNA vaccines and show a trend of promoting sustained enhancement of antibody response, suggesting their potential application value as adjuvants for mRNA vaccines.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a nano-selenium-enhanced rabies mRNA-LNP vaccine, characterized in that, Includes the following steps: Na2SeO3 solution and ascorbic acid solution were mixed and then aged to obtain selenium nanoparticles. A recombinant vector containing the rabies virus glycoprotein G encoding gene was constructed, and the recombinant vector was linearized to obtain a linearized plasmid template; the accession number of the rabies virus glycoprotein G encoding gene is M32751.1; Using the linearized plasmid as a template, mRNA encoding rabies virus glycoprotein G was synthesized through in vitro transcription, capping, and tailing reactions. The citrate buffer containing the SeNPs particles and the rabies virus mRNA was rapidly mixed with the ethanol phase containing lipid components using a microfluidic mixing method to form lipid nanoparticles, which were then purified to obtain a selenium-enhanced rabies mRNA-LNP vaccine.

2. The preparation method according to claim 1, characterized in that, The volume ratio of the Na₂SeO₃ solution to the ascorbic acid solution is 1:1; the concentration of Na₂SeO₃ in the Na₂SeO₃ solution is 10 mM; and the concentration of ascorbic acid in the ascorbic acid solution is 40 mM.

3. The preparation method according to claim 1, characterized in that, The ripening time is 0.5-24 h.

4. The preparation method according to claim 1, characterized in that, The final concentration of the SeNPs particles in the citrate buffer containing the SeNPs particles and the rabies virus mRNA is 10 μg / mL, and the final concentration of the rabies virus mRNA is 100 μg / mL.

5. The preparation method according to claim 1, characterized in that, The lipid components include ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphatidylcholine, cholesterol, and PEG-lipid.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the ionizable lipid, the 1,2-distearate-sn-glycerol-3-phosphatidylcholine, the cholesterol, and the PEG-lipid is 50:10:38.5:1.

5.

7. The preparation method according to claim 1, characterized in that, The volume ratio of the citrate buffer containing the SeNPs particles and the rabies virus mRNA to the ethanol phase containing lipid components is 3:

1.

8. A rabies mRNA-LNP vaccine prepared using the method described in any one of claims 1-7.

9. Application of nano-selenium as an adjuvant in rabies mRNA vaccine.

10. The application according to claim 9, characterized in that, The method for preparing the nano-selenium includes the steps of mixing Na2SeO3 solution and ascorbic acid solution and then aging the mixture to obtain the nano-selenium.

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